Mast Cells and Basophils: Development, Activation and Roles in Allergic Autoimmune Disease (Novartis Foundation Symposium 271)

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Novartis Foundation Symposium 271

MAST CELLS AND BASOPHILS: DEVELOPMENT, ACTIVATION AND ROLES IN ALLERGIC/ AUTOIMMUNE DISEASE

2005


The Novartis Foundation is an international scientific and educational charity (UK Registered Charity No. 313574). Known until September 1997 as the Ciba Foundation, it was established in 1947 by the CIBA company of Basle, which merged with Sandoz in 1996, to form Novartis. The Foundation operates independently in London under English trust law. It was formally opened on 22 June 1949. The Foundation promotes the study and general knowledge of science and in particular encourages international co-operation in scientific research. To this end, it organizes internationally acclaimed meetings (typically eight symposia and allied open meetings and 15–20 discussion meetings each year) and publishes eight books per year featuring the presented papers and discussions from the symposia. Although primarily an operational rather than a grant-making foundation, it awards bursaries to young scientists to attend the symposia and afterwards work with one of the other participants. The Foundation’s headquarters at 41 Portland Place, London W1B 1BN, provide library facilities, open to graduates in science and allied disciplines. Media relations are fostered by regular press conferences and by articles prepared by the Foundation’s Science Writer in Residence. The Foundation offers accommodation and meeting facilities to visiting scientists and their societies.

Information on all Foundation activities can be found at http://www.novartisfound.org.uk

Novartis Foundation Symposium 271


2005

Copyright © 2005

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Participants of Novartis Foundation symposium on Mast cells and basophils: development, activation and roles in allergic/autoimmune disease Standing: Yukihiro Kitamura, Silvia Monticelli, Timothy Williams, Israel Pecht, David Lee, Richard Stevens, Andrew Walls, Don MacGlashan, Shamshad Cockcroft, Dean Metcalfe, Shigeo Koyasu, Peter Bradding, Juan Rivera, Henry Metzger, Toshiaki Kawakami, Jean Marshall, Ehud Razin, Anna Koffer, Hans Oettgen, Gerald Dubois Seated: Anjana Rao, Frank Austen, Jeremy Santa Ono, Susan MacDonald, Yuko Kawakami, Melissa Brown

Contents

Symposium on Mast cells and basophils: development, activation and roles in allergic/ autoimmune disease, held at the Novartis Foundation, London, 16–18 November 2004 Editors: Derek J. Chadwick (Organizer) and Jamie Goode This symposium was based on a proposal made by Santa Jeremy Ono Santa Jeremy Ono

Chair’s introduction

1

Yukihiko Kitamura MITF and SgIGSF: an essential transcription factor and its target adhesion molecule for development and survival of mast cells 4 Discussion 11 Paul J. Bryce, Mendy L. Miller, Ichiro Miyajima, Mindy Tsai, Stephen J. Galli and Hans C. Oettgen Immune sensitization in the skin is enhanced by antigen-independent effects of IgE on mast cells 15 Discussion 24 Yasuko Furumoto, Gregorio Gomez, Claudia Gonzalez-Espinosa, Martina Kovarova, Sandra Odom, John J. Ryan and Juan Rivera The role of Src family kinases in mast cell effector function 39 Discussion 47 Richard L. Stevens, Nasa Morokawa, Jing Wang and Steven A. Krilis RasGRP4 in mast-cell signalling and disease susceptibility 54 Discussion 68 J. Abramson, E. A. Barbu and I. Pecht Regulation of mast cell secretory response to the type I Fce receptor: inhibitory elements and desensitisation 78 Discussion 89 General discussion I

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Toshiaki Kawakami, Jiro Kitaura, Wenbin Xiao and Yuko Kawakami IgE regulation of mast cell survival and function 100 Discussion 108 See-Ying Tam, Janet Kalesnikoff, Susumu Nakae, Mindy Tsai and Stephen J. Galli RabGEF1, a negative regulator of Ras signalling, mast cell activation and skin inflammation 115 Discussion 124 Masako Toda, Takao Nakamura, Masaharu Ohbayashi, Yoshifumi Ikeda, Maria Dawson, Ricardo Micheler Richardson, Andrew Alban, Benjamin Leed, Dai Miyazaki and Santa Jeremy Ono Role of CC chemokines and their receptors in multiple aspects of mast cell biology: comparative protein profiling of FceRI- and/or CCR1-engaged mast cells using protein chip technology 131 Discussion 140 General discussion II

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Shigeo Koyasu, Akiko Minowa, Yasuo Terauchi, Takashi Kadowaki and Satoshi Matsuda The role of phosphoinositide-3-kinase in mast cell homing to the gastrointestinal tract 152 Discussion 161 K. Frank Austen The mast cell and the cysteinyl leukotrienes Discussion 176

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Silvia Monticelli, K. Mark Ansel, Dong U. Lee and Anjana Rao Regulation of gene expression in mast cells: micro-RNA expression and chromatin structural analysis of cytokine genes 179 Discussion 187 Cellina Cohen-Saidon and Ehud Razin The involvement of Bcl-2 in mast cell apoptosis 191 Discussion 195 General discussion III

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P. A. Nigrovic and D. M. Lee arthritis 200 Discussion 210

Mast cells in autoantibody responses and

CONTENTS

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Greg D. Gregory, Allison Bickford, Michaela Robbie-Ryan, Mindy Tanzola and Melissa A. Brown MASTering the immune response: mast cells in autoimmunity 215 Discussion 225 Dean D. Metcalfe Discussion 242

Mastocytosis

Index of contributors Subject index

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Participants

K. Frank Austen Brigham and Women’s Hospital, Harvard Medical School, Division of Rheumatology, Immunology and Allergy, One Jimmy Fund Way, Smith Bldg, Boston, MA 02115, USA Peter Bradding Department of Respiratory Medicine, Glenfield Hospital, Leicester LE3 9QP, UK Melissa Brown Northwestern University School of Medicine, Department of Microbiology and Immunology, Tarry Medical Research Building 7-711, mail code S213, 320 East Superior Street, Chicago, IL 60611-3010, USA Shamshad Cockcroft Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK Gerald Dubois Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK Stephen J. Galli Departments of Pathology and of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA Toshiaki Kawakami La Jolla Institute for Allergy and Immunology, Division of Cell Biology, 10355 Science Center Drive, San Diego, CA 92121, USA Yukihiko Kitamura Developmental Research Laboratories, Shionogi & Co Ltd, Futaba-cho 3-1-1, Toyonaka, Osaka, 561-0825, Japan Anna Koffer Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK Shigeo Koyasu Keio University School of Medicine, Department of Microbiology and Immunology, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan xi

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David M. Lee Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Smith Building, Room 538A, 1 Jimmy Fund Way, Boston, MA 02115, USA Susan M. MacDonald Division of Allergy and Clinical Immunology, Asthma and Allergy Center, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801, USA Donald MacGlashan Johns Hopkins University School of Medicine, Department of Medicine, Clinical Immunology Division, 5501 Bayview Circus, Baltimore, MD 21224, USA Jean S. Marshall Department of Microbiology and Immunology, Dalhousie University, Sir Charles Tupper Medical Building, 7-G Lab, Halifax, Nova Scotia, B3H 1X5, Canada Dean D. Metcalfe NIAID, NIH, Laboratory for Allergic Diseases, 10 Center Drive, MSC 1881, Bldg 10, Rm 11C205, Bethesda, MD 20892-1881, USA Henry Metzger NIAMS, NIH, 9N-228, 10 Center Drive, MSC 1820, Bethesda, MD 20892-1820, USA Silvia Monticelli (Novartis Foundation Bursar) CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02215, USA Hans Oettgen Clinical Director, Division of Immunology, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA Santa J. Ono (Chair) Department of Immunology, Institutes of Ophthalmology and Child Health, University College London, 11-43 Bath Street, London EC1V 9EL, UK Israel Pecht Department of Immunology, The Weizmann Institute of Science, P O Box 26, Rehovot, 76100, Israel Anjana Rao Harvard Medical School and CBR Institute for Biomedical Research, Department of Pathology, 200 Longwood Avenue, Warren Alpert Bldg, Rm 152, Boston, MA 02115, USA Ehud Razin Biochemistry, Hebrew University Medical School, PO Box 12272, Jerusalem 91120, Israel

PARTICIPANTS

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Juan Rivera Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Sciences, Bethesda, MD 20892-1820, USA Richard Stevens Brigham and Women’s Hospital, Department of Medicine, One Jimmy Fund Way, Smith Bldg., Rm 616B, Boston, MA 02115, USA Andrew Walls Immunopharmacology Group, Southampton General Hospital, Level F, South Block, Southampton SO16 6YD, UK Timothy Williams Leukocyte Biology, Biomedical Sciences Division, SAF Building, South Kensington Campus, London, SW7 2AZ, UK

Chair’s introduction Santa Jeremy Ono University College London, 11-43 Bath Street, London EC1V 9EL, UK

I would like to welcome all of you to this Novartis Foundation Symposium on Mast cells: development, activation and roles in allergic/autoimmune disease. We are fortunate to have assembled a tremendous group of investigators in the field and I am sure that this will result in an intense scientific meeting focusing on the latest basic (primarily molecular) research studies on the mast cell and basophil, and their relation to allergic and autoimmune diseases. The overall goal of this symposium is to advance our knowledge of the roles of these two granulocytes in varied disease settings such as those occurring in allergic inflammation and autoimmune disease. With its focus on molecular aspects of mast cells and basophils, the meeting is part of a distinguished history of Novartis Foundation symposia in the area of hypersensitivity and autoimmunity. We all recall the first superb symposium chaired by Henry Metzger from the National Institutes of Health and the more recent one on anaphylaxis chaired by Steve Galli from Stanford. Importantly, this meeting will be quite distinct from the latter: while there is some overlap (since IgE signalling is key to anaphylaxis), the topics covered in that meeting were much broader, with a focus on the disease. This symposium, in contrast, focuses on mast cell development, activation and communications with other cells at the molecular level. The three specific goals of the symposium are: (1) To present recent advances relating to the factors and mechanisms that regulate the growth, differentiation, and function of mast cells and basophils. (2) To discuss new technological advances that directly impact studies on mast cells and basophils. (3) To integrate the basic science findings on mast cells and basophils into the framework of therapeutic potential and treatment of diseases such as allergic inflammation and autoimmune disease which are mediated, in part, by these granulocytes. The meeting will focus on contemporary issues of mast cell/basophil research as they relate to the pathogenesis of allergic and autoimmune diseases. The topics will include: (1) the development of mast cells and basophils; (2) early and late events in IgE/antigen activation of mast cells and basophils; (3) mechanisms of exocytosis; (4) non-IgE mediated activation of mast cells and basophils, as well as those 1

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surface receptors that dampen activation responses; (5) protease, proteoglycan, lipid, and cytokine mediators released from activated mast cells and basophils; and (6) bilateral interactions of mast cells with other cell types. The presentations in the area of mast cell development will examine some of the key intracellular factors that control mast cell development in humans and mice. Analysis of gene expression in mast cells that have been activated by different mechanisms is yielding important new information on genes and their products that are involved in mast cell development and activation. Additional information on embryonic stem cells that differentiate into mast cells is also emerging. Specifically, in vitro differentiated mast cells in adoptive transfer approaches address issues concerning mast cell development, signal transduction and function in vivo. Presentations on early events in FceRI-mediated activation of mast cells and basophils will focus on the molecular basis of activation of these cells. They will include models stemming from biophysical and crystallographic studies of the highaffinity IgE receptor FceRI and its interactions with IgE. Other topics will include major molecules and their interactions that govern the signalling pathways stimulated by FceRI in mast cells. The task ahead is to determine the critical factors that regulate the strength and persistence of signalling. Data on the relationship between ligand valency, affinity, and the kinetics of binding to a variety of cellular responses will be presented. The value of a quantitative model of the signalling cascades initiated by the aggregation of FceRI, as well as some of the difficulties encountered in the development and use of such a model, should also be discussed. The interactions between multivalent antigens and sIgE give rise to a complex distribution of FceRI aggregates on the surfaces of mast cells and basophils. Quantifying the clustering of FceRI in ‘real time’ is yielding new insight into the biophysics of IgEmediated mast cell activation. Other discussions might include the roles of detergent-insoluble membrane microdomains and lipid rafts. This will also include discussion on the facilitation of tyrosine phosphorylation of cross-linked receptors by Lyn in a process that is regulated, in part, by the actin cytoskeleton. Presentations on signalling complexes and downstream signalling in FceRI-mediated activation of mast cells and basophils will explore some of the downstream events that occur prior to exocytosis of the cell’s secretory granules. Discussions will likely focus on IgE receptor-activated macromolecular signalling complexes in mast cells, as well as the importance of the constituent molecules in mast cell degranulation and cytokine production. There will also be presentations and discussion on the exocytosis of mediators from activated mast cells and basophils. They will include talks on cytoskeletal rearrangements required for degranulation of these effector cells, and the link between membrane activation events and these processes. We also anticipate active debate on inhibition of FceRI-mediated responses and non-IgE mechanisms of mast cell and basophil activation focusing on the signalling

CHAIR’S INTRODUCTION

3

pathways controlled by cytokines, chemokines and their receptors. It is now apparent that FceRI-mediated activation of mast cells and basophils can be either stimulated or counteracted by other receptors on the surfaces of these cells. It is also apparent that certain populations of mast cells and basophils can be activated by surface receptors other than FceRI. Of particular interest will be the role chemokine receptors play in mast cell development, mast cell progenitor homing, and mast cell activation. The symposium will end with a discussion of the interaction of mast cells and other cell types, and the role of the mast cells/basophils in disease. Some of the discussions will naturally focus on the role of mast cells in allergic inflammation, and the role of stabilization of these cells in novel therapies. There will also be discussions on mastocytosis and the mutations that give rise to this phenotype. In addition, significant attention will be placed on recent data suggesting a role for mast cells in multiple sclerosis, diabetes and HIV-1 infection. Indeed recent studies have implicated mast cells in processes that control the onset and severity of experimental allergic encephalomyelitis and insulin-dependent diabetes (IDDM) in mice. The strategic location of mast cells in the central nervous system (in multiple sclerosis) and in the pancreas (in IDDM) as well as their ability to express a variety of cytokines and other inflammatory mediators raises the possibility that mast cells either directly initiate the inflammatory responses or act to modulate the character of the T cell response in this disease. In conclusion, I anticipate that this symposium (and the book that results from the presentations and discussions) will take its place alongside the previous Novartis Foundation symposia chronicling the study of mast cells and basophils in health and disease. Let the work and the discussions help form the salient future questions, and draw in young scientists who will carry forward this field of research.

MITF and SgIGSF: an essential transcription factor and its target adhesion molecule for development and survival of mast cells Yukihiko Kitamura Developmental Research Laboratories, Shionogi & Co., Futaba-cho 3-1-1, Toyonaka, Osaka 561-0825, Japan

Abstract. MITF transcription factor is not produced in mutant mice of WBB6F1Mitf mi-vga9/Mitf mi-vga9. When bone marrow cells of normal WBB6F1-+/+ mice were transplanted to the irradiated WBB6F1-KitW/KitW-v mice, which are genetically mast cell deficient, erythrocytes, neutrophils, macrophages, B cells and T cells of the donor origin developed in the recipients. On the other hand, when bone marrow cells of WBB6F1Mitf mi-vga9/Mitf mi-vga9 mice were transplanted to similarly irradiated WBB6F1-KitW/KitW-v mice, erythrocytes, neutrophils, macrophages, B cells and T cells of the donor origin developed but mast cells never appeared. To identify the targets of MITF, we elaborated a subtracted cDNA library between cultured mast cells (CMCs) derived from +/+ mice and CMCs from MITF mutant mice. We obtained a clone encoding SgIGSF, which was expressed on the adhesion surface of +/+ CMCs attaching to fibroblasts. CMCs of WBB6F1-Mitf mi-vga9/ Mitf mi-vga9 mice did not express SgIGSF, did not attach to fibroblasts and did not survive in the peritoneal cavity of WBB6F1-KitW/KitW-v mice. When MITF or SgIGSF cDNA was transfected to WBB6F1-Mitf mi-vga9/Mitf mi-vga9 CMCs, they attached to fibroblasts and showed an improved survival in the peritoneal cavity of WBB6F1-KitW/KitW-v mice. MITF and SgIGSF appeared essential for the development and survival of mast cells in tissues of adult WBB6F1-KitW/KitW-v mice. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 4–14

Three spontaneously occurring mast cell-deficient mutant mice have been reported. We found that mutant mice of W/W v or Sl/Sl d genotype lack mast cells (Kitamura & Go 1979, Kitamura et al 1978). Then the W locus was identified as encoding the KIT receptor tyrosine kinase (hereafter KIT), and the Sl locus as encoding the ligand of KIT (KIT ligand, KITL). Lack of mast cells in connective and mucosal tissues of KitW/KitW-v and Kitl Sl/Kit Sl-d mutant mice indicates the essential role 4

MITF TRANSCRIPTION FACTOR AND SgIGSF ADHESION MOLECULE

5

of KITL–KIT signals for development of both connective tissue-type and mucosal-type mast cells. Microphthalmia (mi ) is the first-found mutant allele at the mi locus. In addition to the small eyes, mice of mi/mi genotype had been known to show depletion of pigment in both hair and eyes and osteopetrosis. Stevens & Loutit (1982) and Stechschulte et al (1987) found a decrease in mast cells and deficiency in natural killer activity in mi/mi mice. The depletion of mast cells in mi/mi mice is not as severe as in the KitW/KitW-v and Kitl Sl/Kitl Sl-d mice. The number of mast cells in the skin tissues of KitW/KitW-v and Kitl Sl/Kitl Sl-d mice was approximately 1% that of normal control (+/+) mice. In contrast, the number of remaining mast cells in the skin of mi/mi mice was approximately 30% that of +/+ mice (Ebi et al 1990). Although the phenotype of the remaining mast cells appeared normal in the skin of KitW/KitW-v and Kitl Sl/Kitl Sl-d mice, the phenotype of mast cells was abnormal in the skin of mi/mi mice. Heparin and histamine content of the mi/mi skin mast cells were estimated to be 34% and 18% of those of +/+ mast cells, respectively (Kasugai et al 1993). Moreover, the deficient expression of the mouse mast cell protease 6 (mMCP-6) gene was demonstrated using in situ hybidization histochemistry. The mi locus encodes a basic helix-loop-helix leucine zipper-type transcription factor (Hodgkinson et al 1993). Hereafter, it is called microphthalmia transcription factor (MITF). Cultured mast cells (CMCs) were obtained from the spleen cells of Mitf mi/Mitf mi mice in the presence of interleukin (IL) 3, and their gene expression phenotypes were compared to those of +/+ CMCs. The expression of genes encoding KIT (Ebi et al 1992, Tsujimura et al 1996), granzyme B, tryptophan hydroxylase (TPH, the key enzyme for the synthesis of serotonin) (Ito et al 1999), mMCP-2 (Ge et al 2001), mMCP-4 (Jippo et al 1999), mMCP-5 (Morii et al 1997), mMCP-6 (Morii et al 1996), mMCP-7 (Ogihara et al 2001) and mMCP-9 was severely impaired in CMCs derived from Mitf mi/Mitf mi mice. The MITF encoded by the mutant Mitf mi allele (hereafter mi-MITF) deletes 1 of 4 arginines in the basic domain (Hodgkinson et al 1993). The normal amount of the abnormal mi-MITF was produced in CMCs derived from Mitf mi/Mitf mi mice. The mutant mi-MITF was defective in DNA-binding potential, and therefore lacked the transcription-promoting activity. On the other hand, MITF itself was not transcribed in CMCs of Mitf mi-vga9/Mitf mi-vga9 mice due to the insertion of a transgene at the promoter region of the MITF gene. The transcription of mMCP-4, mMCP-5 and mMCP-6 genes was severely impaired in both Mitf mi/Mitf mi and Mitf mi-vga9/Mitf mivga9 CMCs, indicating that MITF was essential for their transcription (Ito et al 1999). The transcription of KIT, granzyme B and TPH genes was severely impaired in Mitf mi/Mitf mi CMCs, but moderately impaired in Mitf mi-vga9/Mitf mi-vga9 CMCs. The presence of mi-MITF rather than the absence of MITF inhibited the transcription of KIT, granzyme B and TDH genes. In other words, the mi-MITF appeared to inhibit the transcription-promoting activity of other transcription factors (Ito et al

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1999). The Mitf mi is an inhibitory mutation (Morii et al 2001a), and the Mitf mi-vga9 is a null mutation (Morii et al 2001b). The SgIGSF adhesion molecule The number of mast cells decreased, but an appreciable number of mast cells remained in the skin of Mitf mi/Mitf mi and Mitf mi-vga9/Mitf mi-vga9 mice. Mitf mi/Mitf mi mice die on weaning because teeth do not erupt due to the severe osteopetrosis, whereas most Mitf mi-vga9/Mitf mi-vga9 mice grow to adulthood. The severe osteopetrosis of Mitf mi/Mitf mi mice may be attributable to defective differentiation of osteoclasts by the inhibitory effect of the mutant mi-MITF (Steingrimsson et al 2002). Mast cells did not develop in tissues other than the skin of Mitf mi-vga9/Mitf mi-vga9 mice throughout our observation period (Jippo et al 2003). Since mast cells are absent in the peritoneal cavity of Mitf mi/Mitf mi mice, CMCs obtained from the haematopoietic cells of Mitf mi/Mitf mi and Mitf mi-vga9/Mitf mi-vga9 were injected into the peritoneal cavity of Mitf mi-vga9/Mitf mi-vga9 mice. These CMCs from MITF mutant mice disappeared within 5 weeks after the injection, whereas the control +/+ CMCs survived. The poor survival of CMCs of MITF mutant mice was reproducible in vitro. +/+ CMCs survived on the monolayer of NIH/3T3 fibroblasts, but Mitf mi/Mitf mi or Mitf mi-vga9/Mitf mi-vga9 CMCs did not. Moreover, +/+ CMCs adhered to NIH/3T3 fibroblasts, but Mitf mi/Mitf mi and Mitf mi-vga9/Mitf mi-vga9 CMCs did not (Ito et al 2003). From a cDNA library of +/+ CMCs, we subtracted mRNAs expressing Mitf mi/ Mitf mi CMCs and found a clone encoding SgIGSF, a recently identified member of the immunoglobulin superfamily (Ito et al 2003). Northern blot analysis revealed that SgIGSF was expressed by +/+ CMCs but not by CMCs derived from MITF mutant mice. Immunohistochemical analysis showed that SgIGSF localized to the cell–cell contact areas between +/+ CMCs and NIH/3T3 fibroblasts. NIH/3T3 fibroblasts did not express SgIGSF, indicating that SgIGSF acted as a heterophilic adhesion molecule. The ligand of SgIGSF expressed on the fibroblasts remains to be identified. Transfection of Mitf mi/Mitf mi or Mitf mi-vga9/Mitf mi-vga9 CMCs with SgIGSF cDNA normalized their adhesion to NIH/3T3 fibroblasts. Transfection of Mitf mi-vga9/ Mitf mi-vga9 CMCs with normal MITF cDNA elevated their SgIGSF expression to normal levels. Transfection of SgIGSF or MITF cDNA improved the survival of Mitf mi-vga9/Mitf mi-vga9 CMCs in the peritoneal cavity of Mitf mi-vga9/Mitf mi-vga9 or KitW/KitW-v mice (Ito et al 2003). SgIGSF mediated the adhesion of CMCs to fibroblasts and the transcription of SgIGSF was critically regulated by MITF. The cause of mast cell deficiency in MITF mutant mice We produced Mitf mi-vga9/Mitf mi-vga9 and KitW/KitW-v mice in the same genetic background. WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice are suitable for in vivo studies on effects


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of MITF on mast cell development. Since MITF is expressed in both mast cells and tissues where mast cells develop, there is a possibility that the Mitf mi-vga9/Mitf mi-vga9 mice may show abnormalities in both mast cell precursors and tissue environments. This possibility was examined by bone marrow and skin transplantation (Morii et al 2004). Bone marrow cells of B6-Mitf mi-vga9/Mitf mi-vga9 mice were transplanted to irradiated WBB6F1-KitW/KitW-v mice that possessed normal tissue environment. Erythrocytes, neutrophils, macrophages, B cells and T cells of B6-Mitf mi-vga9/ Mitf mi-vga9 mouse origin develop in the WBB6F1-KitW/KitW-v recipients, but mast cells did not develop in all tissues including skin. Moreover, the number of developing mast cells in the skin tissues of WBB6F1-KitW/KitW-v mice was much lower when grafted to WBB6F1-Mitf mi-vga9/Mitf mi-vga9 recipients than when grafted to WBB6F1+/+ recipients. These results indicated that mast cell precursors of WBB6F1Mitf mi-vga9/Mitf mi-vga9 mice were defective. As already mentioned, the donor B6-Mitf mi-vga9/Mitf mi-vga9 mice had a decreased but recognizable number of skin mast cells that had developed in the embryonic age (Kitamura et al 1979). In spite of the presence of mast cells in the skin tissue of the donor B6-Mitf mi-vga9/Mitf mi-vga9 mice, their bone marrow stem cells did not differentiate to mast cells in tissues of irradiated WBB6F1-KitW/KitW-v adult mice. The mechanisms controlling mast cell development in embryonic skin appeared different from those of adult skin. The expression of the KIT gene was deficient in CMCs of Mitf mi-vga9/Mitf mi-vga9 genotype, and their response to KITL was significantly lower when compared to the response of +/+ CMCs (Morii et al 2004). The transcription of SgIGSF was severely impaired in Mitf mi-vga9/Mitf mi-vga9 CMCs. The deficient differentiation of Mitf mi-vga9/Mitf mi-vga9 stem cells to mast cells may, at least in part, be attributable to the impaired expression of KIT and SgIGSF. When bone marrow cells of WBB6F1-+/+ mice were transplanted, the number of developing mast cells was significantly lower in examined tissues of WBB6F1Mitf mi-vga9/Mitf mi-vga9 recipients than in those of WBB6F1-KitW/KitW-v recipients. The deficient development of +/+ mast cells was more severe in the mesentery and spleen of Mitf mi-vga9/Mitf mi-vga9 mice than in their stomach. Almost no mast cells were detectable in the mesentery and spleen of WBB6F1-Mitf mi.vga9/Mitf mi-vga9 mice after the bone marrow transplantation (Morii et al 2004). We also compared the change of mast cell number in skin grafts transplanted from WBB6F1-KitW/KitW-v mice or from WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice to the back of WBB6F1-+/+ mice (Morii et al 2004). Skin grafts from WBB6F1KitW/KitW-v mice did not contain mast cells before the transplantation, but the mast cell number in grafts remarkably increased after transplantation to WBB6F1-+/+ mice. Although the mast cell number gradually increased in skin tissues grafted from WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice, the magnitude of increase was significantly lower than that observed in skin tissues grafted from WBB6F1-KitW/KitW-v mice. These

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results suggested that the tissue environment for mast cell development was defective in WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice. MITF appeared essential for the function of both mast cell precursors and tissue environments for their development. In addition to deficient transcription of KIT and SgIGSF, which may explain the defect of mast cell precursors, transcription of molecules regulating development of mast cells may be deficient in tissues of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice. Reverse transcriptase PCR results using whole tissues of mesentery and spleen of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 and WBB6F1-KitW/KitW-v mice suggested that the expression level of KITL appeared to be comparable between the mesentery and spleen of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 and WBB6F1-KitW/KitW-v mice. Therefore, KITL is not considered to be such a regulating molecule (Jippo et al 2003). WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice may be useful for identifying new microenvironmental factors other than KITL that regulate development of mast cells. Deficient chemotaxis promoting activity of +/+ mast cells in tissues of MITF mutant mice Mast cells play an important role for the innate immunity against acute peritonitis induced by bacterial infection. Echtnacher et al (1996) and Malaviya et al (1996) demonstrated it using WBB6F1-KitW/KitW-v mice. Echtnacher et al (1996) induced acute peritonitis by ligation and puncture of caecum, and Malaviya et al (1996) induced it by the intraperitoneal injection of enterobacteria. The proportion of survival after induction of acute peritonitis was significantly lower in WBB6F1KitW/KitW-v mice than in WBB6F1-+/+ mice. Since WBB6F1-Mitf mi-vga9/Mitf mi-vga9 also lack peritoneal mast cells, we compared the effect of caecal ligation and puncture among WBB6F1-Mitf mi-vga9/Mitf mi-vga9, WBB6F1-KitW/KitW-v and WBB6F1-+/+ mice. The proportion of survival was significantly lower in WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice than in WBB6F1-+/+ mice as in the case of WBB6F1-KitW/KitW-v mice ( Jippo et al 2003). The poor survival of WBB6F1-KitW/KitW-v and WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice was attributed to the deficient influx of neutrophils into the peritoneal cavity. The injection of CMCs derived from WBB6F1-+/+ mice normalized the neutrophil influx and reduced survival rate in WBB6F1-KitW/KitW-v mice, but not in WBB6F1Mitf mi-vga9/Mitf mi-vga9 mice. This was not attributable to a defect of neutrophils of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice because of the following two reasons: (1) injection of tumour necrosis factor (TNF) a increased the neutrophil influx and survival in both WBB6F1-KitW/KitW-v and WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice ( Jippo et al 2003), and (2) WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice which received irradiation and bone marrow transplantation from WBB6F1-+/+ mice also showed poor survival after caecal ligation and puncture in spite of the +/+ donor origin of neutrophils (Morii et al 2004).


9

Although the intraperitoneal injection of WBB6F1-+/+ CMCs normalized the number of mast cells in both the peritoneal cavity and mesentery of WBB6F1KitW/KitW-v mice, it normalized the number of mast cells only in the peritoneal cavity of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice. Mast cells within the mesentery or mast cells in the vicinity of blood vessels appeared to play an important role against acute bacterial peritonitis. Tissues of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice showed a defect not only for +/+ neutrophil infiltration but also for +/+ eosinophil infiltration (Oboki et al 2004). The substance P (SP)-induced eosinophil infiltration was compared between WBB6F1KitW/KitW-v and WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice using air-bleb assay. The air-bleb membrane was composed of the subcutaneous connective tissue. Although the dermis of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice contained 30% of mast cells compared to that of WBB6F1-+/+ mice, the air-bleb membranes formed in the back of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice contained no mast cells. The WBB6F1-Mitf mivga9 /Mitf mi-vga9 mice showed impaired SP-induced eosinophil infiltration as observed in WBB6F1-KitW/KitW-v mice, indicating that mast cells detected in the dermis of WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice did not help SP-induced eosinophil infiltration. Subcutaneous transplantation of CMCs from WBB6F1-+/+ mice normalized SP-induced eosinophil infiltration in WBB6F1-KitW/KitW-v mice but not in WBB6F1Mitf mi-vga9/Mitf mi-vga9 mice. The greater number and the more dispersed distribution pattern of mast cells that appeared in the subcutaneous connective tissue of WBB6F1-KitW/KitW-v mice after the transplantation appeared to explain the difference between WBB6F1-KitW/KitW-v and WBB6F1-Mitf mi-vga9/Mitf mi-vga9 mice. WBB6F1Mitf mi-vga9/Mitf mi-vga9 mice may be useful for studying the effect of anatomical distribution of mast cells on induction of both neutrophil and esosinophil infiltration. Other transcription factors involved in mast cell development Using the GATA-1low mice with a targeted deletion of upstream enhancer and promoter sequences of the Gata1 gene, Migliaccio et al (2003) showed that the reduced expression of Gata1 gene resulted in amplification of the mast cell progenitor compartment, increased apoptotic rate at the precursor level and defective differentiation of the mature mast cells. The expression of both PU.1 and GATA-2 at appropriate levels appears to be required for development of mast cells. Walsh et al (2002) showed that PU.1 antagonized GATA-2 expression during macrophage differentiation, but functioned cooperatively with GATA-2 to specify the mast cell fate. They proposed a developmental model in which reciprocal antagonism between PU.1 and GATA family transcription factors was essential for the differentiation of macrophages, whereas cooperative interaction between PU.1 and GATA-2 was required for the differentiation of mast cells.

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GATA-1, GATA-2, PU.1 and MITF are involved in development of mast cells. GATA-1, GATA-2 and PU.1 appear to be involved in the relatively early stage of development that determines the direction of differentiation. On the other hand, MITF appears to be involved in the relatively late stage; migration of committed precursors, their phenotypic expression within tissues, and survival of differentiated mast cells. References Ebi Y, Kasugai T, Seino Y, Onoue H, Kanemoto T, Kitamura Y 1990 Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts. Blood 75:1247–1251 Ebi Y, Kanakura Y, Jippo-Kanemoto T et al 1992 Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit. Blood 80:1454–1462 Echtenacher B, Mannel DN, Hultner L 1996 Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381:75–77 Ge Y, Jippo T, Lee YM, Adachi S, Kitamura Y 2001 Independent influence of strain difference and mi transcription factor (MITF) on the expression of mouse mast cell chymase. Am J Pathol 158:281–292 Hodgkinson CA, Moore KJ, Nakayama A et al 1993 Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix zipper protein. Cell 74:395–404 Ito A, Morii E, Kim DK et al 1999 Inhibitory effect of the transcription factor encoded by the mi mutant allele in cultured mast cells of mice. Blood 93:1189–1196 Ito A, Jippo T, Wakayama Y et al 2003 SgIGSF: a new mast-cell adhesion molecule used for attachment to fibroblasts and transcriptionally regulated by MITF. Blood 101:2601–2608 Jippo T, Lee YM, Katsu Y et al 1999 Deficient transcription of mouse mast cell protease 4 gene in mutant mice of mi/mi genotype. Blood 93:1942–1950 Jippo T, Morii E, Ito A, Kitamura Y 2003 Effect of anatomical distribution of mast cells on their defense against bacterial infections: demonstration using partially mast cell-deficient tg/tg mice. J Exp Med 197:1417–1425 Kasugai T, Oguri K, Jippo-Kanemoo T et al 1993 Deficient differentiation of mast cells in the skin of mi/mi mice: usefulness of in situ hybridization for evaluation of mast cell phenotype. J Am Pathol 143:1337–1347 Kitamura Y, Go S 1979 Decreased production of mast cells in Sl/Sl d anemic mice. Blood 53:492–497 Kitamura Y, Go S, Hatanaka K 1978 Decrease of mast cells in W/W v mice and their increase by bone marrow transplantaion. Blood 52:447–452 Kitamura Y, Shimada M, Go S 1979 Presence of mast cell precursors in fetal liver of mice. Dev Biol 70:510–514 Malaviya R, Ikeda T, Ross E, Abraham SN 1996 Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 381:77–80 Migliaccio AR, Rana RA, Sanchez M et al 2003 GATA-1 as a regulator of mast cell differentiation revealed by the phenotype of the GATA-1-low mouse mutant. J Exp Med 197:281–296 Morii E, Tsujimura T, Jippo T et al 1996 Regulation of mouse mast cell protease 6 gene expression by transcription factor encoded by mi locus. Blood 88:2488–2494 Morii E, Jippo T, Tsujimura T et al 1997 Abnormal expression of mouse mast cell protease 5 gene in cultured mast cells derived from mutant mi/mi mice. Blood 90:2601–2608


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Morii E, Oboki K, Ishihara K, Jippo T, Hirano Y, Kitamura Y 2004 Roles of MITF for development of mast cells in mice: effects on both precursors and tissue environments. Blood 104:1656–1661 Morii E, Ogihara H, Oboki K et al 2001a Inhibitory effect of MITF encoded by mutant mi allele on GABP-mediated transcript expression in mouse mast cells. Blood 97:3032– 3039 Morii E, Ogihara H, Kim DK et al 2001b Importance of leucine zipper domain of mi transcription factor (MITF) for differentiation of mast cells demonstrated using mi ce/mi ce mutant mice of which MITF lacks the zipper domain. Blood 97:2035–2044 Oboki K, Morii E, Kitamura Y 2004 Deficient eosinophil chemotaxis promoting activity of genetically normal mast cells transplanted into subcutaneous tissue of Mitf mi-vga9/Mitf mi-vga9 mice: comparison of the activity and mast cell distribution pattern with KitW/KitW-v mice. Am J Pathol 165:1141–1150 Ogihara H, Morii E, Kim DK, Oboki K, Kitamura Y 2001 Inhibitory effect of transcription factor encoded by mi microphthalmia allele on transactivation of mouse mast cell protease 7 gene. Blood 97:645–651 Stechschulte DJ, Sharma R, Dileepan KN et al 1987 Effect of the mi allele on mast cells, basophils, natural killer cells, and osteoclasts in C57BL/6 mice. J Cell Physiol 132:565–570 Steingrimsson E, Tessarollo L, Pathak B et al 2002 Mitfand Tfe3, two members of Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development. Proc Natl Acad Sci USA 99:4477– 4482 Stevens J, Loutit JF 1982 Mast cells in spotted mutant mice (W Ph mi ). Proc R Soc London B Biol Sci 215:405– 409 Tsujimura T, Morii E, Nozaki M et al 1996 Involvement of transcription factor encoded by the mi locus in the expression of c-kit receptor tyrosine kinase in cultured mast cells of mice. Blood 88:1225–1233 Walsh JC, DeKoter RP, Lee HJ et al 2002 Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity 17:665–676

DISCUSSION Stevens: We believe mouse mast cell protease (mMCP) 6 is one of the primary factors exocytosed from activated mast cells that regulates neutrophil extravasation in tissues (Huang et al 1998). In contrast, others feel the most important factor is the cytokine TNFa (Echtenacher et al 1996). You showed that the mast cellmediated recruitment of neutrophils into tissues is greatly diminished in tg/tg mice which have a defect in the expression of the transcription factor MITF. You also showed that the levels of mMCP-6 protein and mRNA are quite low in tg/tg mouse mast cells relative to normal mast cells. These data are consistent with a prominent role for mMCP-6 in neutrophil extravasation. Nevertheless, if you activate tg/tg mouse mast cells via calcium ionophore (or with IgE followed by antigen), do the treated cells generate normal amounts of TNFa? Kitamura: I haven’t done this experiment. Koyasu: When you isolated SgIGSF, you did this by comparing the cultured mast cells from mi/mi mice and the wild-type mice. But how many genes are differentially activated altogether?

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Kitamura: We screened more than 500 clones. Only one of these 500 clones encoded an adhesion molecule, SgIGSF. Rao: Can we start at the beginning? You said that the mi/mi mice have the transcription factor completely knocked out. Is that correct? What about the tg/tg mice? Kitamura: The tg/tg mice did not express any MITF, but mi/mi mice expressed the inhibitory MITF. This inhibited the transcription of target genes by other transcription factors. The tg/tg mice were produced by the insertion of a transgene into the promoter region of MITF gene. Rao: Is that a hypomorph (with a lower level of activity), or a complete loss of the transcription factor? Kitamura: It’s a complete loss, at least in cultured mast cells. Galli: You showed that when +/+ skin is transplanted onto the W/W v mouse, the number of mast cells in the skin is doubled. Can you speculate about the mechanism? Kitamura: The skin graft is transplanted onto the back, and there may be some kind of stimulation. I do not exactly know what kind of cells may be involved in the stimulation. For example, some cells such as fibroblasts may produce stem cell factor. Galli: If you sacrifice such mice many months after the skin transplantation, is the high level of dermal mast cells maintained? Kitamura: I don’t know. I examined it only 10 weeks after. Ono: Have you looked at a system where pathogen is injected at different positions in the body and you then subsequently examine the closest draining lymph node to see whether you have a similar induction of lymph node enlargement in your mice? Or you could do this by allergen challenge at different sites? This may reveal variation in responses in different tissues and also of different types of mast cells. Kitamura: No, we haven’t done this. Koyasu: In your tg/tg mice, do you still have some mast cells in the skin? Kitamura: Yes. Koyasu: When you take bone marrow cells from tg/tg and transplant them to W/W v mice, do you see any mast cell development? Kitamura: No. Koyasu: When you transplant wild-type bone marrow cells to tg/tg mice, do you get mast cells in the skin? Kitamura: Yes. Koyasu: Your precursors can develop into dermal mast cells in the environment of tg/tg—those mice have dermal mast cells—yet if you transplant bone marrow cells from tg/tg into W/W v mice, you don’t get any. How do you explain this? Kitamura: In tg/tg mice the situation is rather complicated. Mast cells develop in embryonic age only in the dermis. In other tissues mast cells develop after birth.


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The mechanism controlling mast cell development in embryos may differ from the mechanism controlling mast cell development in adults. In any tissues of adult W/W v mice, tg/tg stem cells cannot differentiate into mast cells. Koyasu: Is that because of the environment or is it still the precursors? For example, if you transplant fetal liver cells from tg/tg mice, what happens? Kitamura: I don’t know. The mast cell deficiency is mostly because of the defect of mast cell precursors themselves, although there may be an environmental influence. Galli: I have a question about the adhesion of mast cells to fibroblasts. Your work and that of others also showed that KIT/SCF interactions can contribute to the adhesion. Can you tell us about the relative importance, in mast cell-fibroblast adhesion, of sgIGSF versus KIT, versus laminin and so on? Kitamura: In experiments with NIH-3T3 fibroblasts and cultured mast cells, KIT/SCF is probably more important. We also did experiments using the mesothelial cells of the peritoneum and mast cells. In this case SgIGSF is more important. Galli: Is there any role for KIT/SCF in mast cell-peritoneal mesothelial cell interactions? Kitamura: Other factors may be involved, but the two most important factors are KIT/SCF and SgIGSF/its ligand, in the interaction of mast cells and fibroblasts. In the case of the mesothelial cells, SgIGSF/its ligand is more important. Razin: What do we know about the role played by SgIGSF in osteoclasts, melanocytes and other cells that express MITF? Kitamura: I don’t know whether osteoclasts express SgIGSF. Razin: What about the role of SgIGSF in melanocytes? Kitamura: I don’t know. Kawakami: Is adhesion in mi/mi or tg/tg mice generally defective? Kitamura: SgIGSF was ubiquitously expressed in many kinds of cells. In the mi/mi mutant, only the mast cells don’t express SgIGSF, so we conclude that only in mast cells does mi control the expression of the SgIGSF. Kawakami: So it sounds like adhesion to fibronectin is intact. Kitamura: I’m not sure about this. Stevens: Different isoforms of the transcription factor MITF have been isolated and characterized (Takemoto et al 2002). Is the reason why some mast cells are present in the skin of tg/tg mice because a different promoter and/or splicing event is used to control the expression of a particular MITF isoform in this tissue? In this regard, have you carried out in situ hybridization or RT-PCR analyses to evaluate the steady-state levels of MITF mRNA in cutaneous mast cells, as well as to identify its primary isoform? Kitamura: I don’t think so, although it is possible.

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Razin: In the original paper published in Cell (Hodgkinson et al 1993) the group that produced the tg/tg mice did in situ hybridization. They found a bit in the uterus and some in the eye (only at the level of the transcript). Rao: Is there an alternative promoter? Razin: The cassette was inserted upstream of exon 1. The question is whether there is any promoter upstream of exon 1. Rao: There is an alternative exon 1 that might be used as a different promoter. Razin: People have characterized about five alternatively spliced forms of MITF. Koyasu: When you have the mi/mi locus, you said that this product is inhibitory. This strongly suggests that there must be an important partner for MITF. What is known about the combination of MITF with other transcription factors? Kitamura: For example, the mutant mi-MITF inhibited GABP-mediated transcription. As a result, the content of heparin is deficient in skin mast cells of mi/mi mice. Oettgen: What is the transgene Tg that has been inserted into the promoter to disrupt its function? Is this transgene itself expressed in mast cells? Kitamura: I don’t know the precise molecular mechanism. The mi gene was identified because the transgene was incidentally inserted into the promoter region of the mi gene. Until then the mi locus wasn’t localized. Oettgen: Do you know what the transgene encodes? Kitamura: No. References Echtenacher B, Mannel DN, Hultner L 1996 Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381:75–77 Hodgkinson CA, Moore KJ, Nakayama A et al 1993 Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell 74:395–404 Huang C, Friend DS, Qiu WT et al 1998 Induction of a selective and persistent extravasation of neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6. J Immunol 160:1910–1919 Takemoto CM, Yoon YJ, Fisher DE 2002 The identification and functional characterization of a novel mast cell isoform of the microphthalmia-associated transcription factor. J Biol Chem 277:30244–30252

Immune sensitization in the skin is enhanced by antigen-independent effects of IgE on mast cells Paul J. Bryce, Mendy L. Miller, Ichiro Miyajima*, Mindy Tsai†, Stephen J. Galli† and Hans C. Oettgen1 Division of Immunology, Children’s Hospital, Boston, MA 02115, USA, * First Department of Pediatrics, Kurume University School of Medicine, Kurume, Japan and †Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA

Abstract. Contact sensitivity responses require both effective immune sensitization following cutaneous exposure to chemical haptens and antigen-specific elicitation of inflammation upon subsequent hapten challenge. We have observed that that antigen-independent effects of immunoglobulin E (IgE) antibodies promote immune sensitization to haptens in the skin. Contact sensitivity is markedly impaired in IgE-/- mice but can be restored by either transfer of sensitized cells from wild-type mice or administration of haptenirrelevant IgE before sensitization. Moreover, IgE-/- mice exhibit impairment in the emigration of dendritic cells from the epidermis after hapten exposure. Monomeric IgE has been reported to influence mast cell function. We observe diminished contact sensitivity in mice lacking FceRI or mast cells, and mRNA for several mast cell-associated genes is reduced in IgE-/- vs. wild-type skin after hapten exposure. We propose that levels of IgE normally present in mice favour immune sensitization via antigen-independent effects on mast cells. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 15–38

Classic Type I immediate hypersensitivity reactions arise when allergen specific immunoglobulin E (IgE), bound to mast cells via FceRI, interacts with polyvalent antigen, triggering mast cell activation. Such antigen-specific IgE-driven mast cell activation underlies many forms of acute clinical hypersensitivity responses, including anaphylaxis, food reactions and allergen-induced bronchoconstriction. The development of anti-IgE therapeutics has been driven by evidence that interfering with antigen-induced mast cell activation might attenuate such reactions.

1

This paper was presented at the symposium by Hans C. Oettgen to whom correspondence should be addressed. 15

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In the past few years it has become clear that IgE can exert some receptormediated effects even in the absence of known specific antigen. In cultured mast cells, for instance, monomeric IgE antibodies have been reported to regulate cell growth, protein tyrosine phosphorylation, Ca2+ fluxes, cytokine transcription and mediator release. We have recently investigated whether IgE might exert analogous antigen-independent influences on mast cells in vivo. This report will briefly review the published evidence concerning antigen-independent effects of IgE, as well as provide a detailed presentation of our own observations concerning antigen-independent effects of IgE in vivo. The implications of these findings with respect to cutaneous immune responses will be discussed. Several years ago, analyses of IgE-/- mice generated by gene targeting revealed attenuated IgE receptor expression (both CD23 and FceRI) in the absence of IgE. Our findings demonstrated that IgE, in the absence of antigen, regulates the cell surface expression of CD23 and FceRI, both in vitro and in vivo (Kisselgof & Oettgen 1998, Yamaguchi et al 1997, Yamaguchi et al 1999). This has functional implications, in that the up-regulation of FceRI surface expression on mast cells by IgE enhances serotonin release and cytokine production following subsequent challenge with antigen-specific IgE and relevant antigen (Yamaguchi et al 1997). Basophils from patients treated with the anti-IgE monoclonal antibody, omalizumab, exhibit diminished surface expression of FceRI and decreased allergen responsiveness (Beck et al 2004). In back-to-back reports appearing in 2001, the groups of Krystal and Kawakami reported that monomeric IgE could function as a survival factor for mast cells (Kalesnikoff et al 2001, Asai et al 2001). Moreover, certain IgE antibodies, signalling via FceRI, could trigger mast cell cytokine production in vitro, again in the absence of exposure to known specific antigen (Kalesnikoff et al 2001). Subsequent studies revealed that monoclonal IgE antibodies produced by different hybridomas varied in their capacity to elicit antigen-independent production of cytokines and other mediators by mast cells (Kitaura et al 2003). Some, including the anti-DNP clone SPE-7, were particularly good inducers of cytokine secretion and were dubbed ‘cytokinergic’. In rat basophilic leukaemia cells, this IgE mAb has been reported to lead to increased cytosolic Ca2+ levels and nuclear translocation of NFAT (Pandey et al 2004). A clue to the mechanism of antigen-independent IgE signalling via FceRI was provided in the Kitura et al (2003) study. The investigators observed that monomeric hapten (DNP-lysine) inhibited interleukin (IL) 6 secretion by SPE-7stimulated bone marrow cultured mast cells (BMCMCs), a finding which strongly implicated the antigen-binding site of IgE in the initiation of signalling by monovalent IgE. A possible explanation for this observation was provided by concurrent work published by James and colleagues. They observed that SPE-7 can exist in two isoforms, one which binds to DNP and another which binds to an unrelated protein

IgE ANTIBODIES ENHANCE CONTACT SENSITIVITY RESPONSES

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antigen, thioredoxin, identified by screening a peptide library ( James et al 2003). Taken together with Kitura et al (2003)’s observation regarding inhibition of signalling by monovalent hapten, this finding suggested the possibility that ‘antigenindependent’ signalling might actually be initiated by the interaction of IgE antigen-binding sites with alternative and perhaps even autologous molecules. Alternatively, other mechanisms that also can result in achieving aggregation of FceRI in the absence of specific antigen may contribute to the elicitation of biological responses in mast cells exposed to IgE. We had previously generated IgE-/- mice by gene targeting (Oettgen et al 1994) and observed effects of a lack of IgE on IgE receptor expression. The recent findings of effects of monomeric IgE on cultured mast cells prompted us to compare mast cell-dependent functions in IgE-/- animals and wild-type controls to look for evidence of additional IgE influences on mast cell homeostasis and/or phenotype in vivo. As mast cells had previously been implicated by others in the induction of certain examples of contact sensitivity responses, we chose this as an initial system for examination of IgE–mast cell interactions. Although dermal mast cell numbers in IgE-/- mice and the BALB/c controls were the same, we observed that IgE, independently of exposure to known specific antigen, was required for optimal contact sensitivity responses (Bryce et al 2004). IgE-/- and wild-type BALB/c mice were sensitized epicutaneously on the abdomen with hapten and challenged 5 days later by application of the hapten to the ear (Fig. 1). The ears of BALB/c control mice exhibited abundant infiltrating inflammatory cells following hapten challenge (Fig. 1). The cellular infiltrate consisted mainly of mononuclear cells, neutrophils and some eosinophils, and was associated with substantial dermal oedema. Occasional intraepithelial microabscesses containing neutrophils were also observed. In contrast, contact sensitivity responses in the ears of IgE-/- mice exhibited only a slight cellular infiltration, consisting predominantly of mononuclear cells and occasional eosinophils, but no microabscesses. IgE-/- animals that received IgE (10 mg, i.v.) ~18 hours prior to sensitization demonstrated dermal leukocyte infiltration similar to that observed in wild-type animals, although no intraepithelial abscesses were observed (not shown, see Bryce et al 2004). The same dramatic difference in the expression of contact sensitivity reactions between wild-type BALB/c and IgE-/- mice was observed when ear thickness measurements were used to follow the reaction. Sensitized wild-type animals displayed a robust increase in ear thickness to oxazolone challenge peaking at 24 h (Fig. 2). In contrast, IgE-/- animals developed minimal increases in thickness. In rodents, mast cells are the only dermal cells that express IgE receptors. Mast celldeficient WBB6F1/J-KitW/KitW-v mice also had a significantly impaired ear swelling responses when tested with this protocol compared to wild-type controls (Fig. 2). This defect could be repaired by reconstitution of the mast cell defect by i.v.

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BRYCE ET AL Ox challenge

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FIG. 1. Histological examination of contact sensitivity responses. Oxazolone contact sensitivity protocol and haematoxylin and eosin stained ear sections following oxazolone sensitization and oxazolone or ethanol (EtOH; control) challenge in BALB/c (WT) and IgE-deficient (IgE-/- ) mice. Representative sections are shown from one of four individual animals that were examined per group.

injection of BMCMCs from wild-type (i.e. WBB6F1/J-Kit +/+ ) donors. Parallel studies examining the responses of mice lacking either the FceRI (by virtue of a targeted mutation of the common g -signalling chain shared by FceRI and Fcg RIII) or the Fcg RIII-a chain, revealed that FceRI function had to be intact for expression of contact sensitivity in our oxazolone model. Cellular adoptive transfer and IgE-reconstitution experiments established that IgE antibodies are critical—but in an antigen-irrelevant fashion—during the sensitization phase (but not the elicitation phase) of the contact sensitivity response. Transfer of splenocytes from sensitized wild-type animals conferred robust oxazolone-specific contact sensitivity to both naïve wild-type and IgE-/- recipients (Fig. 3). In contrast, cells from oxazolone-exposed IgE-/- mice were unable to transfer oxazolone sensitivity into either wild-type or IgE-/- recipients. In passive IgE reconstitution experiments, i.v. administration of IgE prior to sensitization enhanced the ability of IgE-/- animals to express contact sensitivity responses to oxazolone in a dose-dependent manner. Injection of IgE after sensitization and prior to challenge had no effect. Reconstitution of serum levels of IgE lower than or equal to those observed in wild-type mice in our colony was sufficient to restore the responses to hapten. Notably, reconstitution of the contact sensitivity response by exogenous IgE in IgE-/- mice did not require known antigenic specificity for the sensitizing hapten.

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Time (h) FIG. 2. IgE- and mast cell-deficient mice have impaired contact sensitivity responses to chemical haptens. Wild-type (WT, = BALB/c), IgE-deficient (IgE-/-) or WBB6F1/J-+/+ or WBB6F1/J-KitW/KitW-v mast cell-deficient (W/W v ) mice were sensitized to oxazolone and challenged 5 days later and ear thickness measured over time. One group of mast cell-deficient mice received an i.v. injection of 10 ¥ 106 wild-type (WBB6F1/J-Kit +/+) BMCMCs (+/+BMCMCs Æ W/W v ) 20 weeks before assessment of contact sensitivity.

BRYCE ET AL OX

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Time (h)

FIG. 3. IgE is required for the optimal expression of the sensitization phase of contact sensitivity to oxazolone. Groups of wild-type or IgE-/- mice were sensitized to oxazolone. Five days later splenocytes were transferred i.v. to naïve recipients (107 cells/animal). Recipients were challenged with oxazolone and mean ear swelling measured. Results are expressed as mean ± SEM (n = 10–12/group).

We observed that each of a panel of four IgE monoclonal antibodies specific for TNP, DNP or ovalbumin was effective in reconstituting the ability to express contact sensitivity responses to oxazolone when given prior to sensitization, whereas a mouse anti-TNP IgG antibody had no detectable effect. Thus the presence of IgE per se is permissive of sensitization to chemical haptens in a manner that is independent of the known antigen specificity of the antibody. Contact sensitizers are potent inducers of dendritic cell emigration from the skin and mice with defective migration of dendritic cells exhibit diminished contact sensitivity responses. Whereas application of oxazolone to the skin of wild-type BALB/c controls led to a reduction of dendritic cells in the epidermis, presumably because of emigration to regional lymph nodes, epidermal dendritic cell numbers were not altered in IgE-/- mice. Mast cells are known to produce cytokines or chemokines (including tumour necrosis factor [TNF]a, IL6, IL1b and MCP-1) critical for the activation of antigen exposed dendritic cells and monomeric IgE signalling has been implicated in the production of some of these same cytokines in BMCMCs. We therefore considered the possibility that the production of such mediators might be dysregulated in the absence of IgE and evaluated dermal levels of these mediators in IgE-/- mice. Oxazolone-induced mRNA levels of IL1b, IL6, MCP-1 and TNFa were all significantly lower in the skin of IgE-/animals than in the wild-type mice (Fig. 4). Restoration of IgE levels by i.v. administration of the ‘cytokinergic’ SPE-7 IgE monoclonal antibodies 18 h before the mice were sacrificed resulted in dose-related increases in the levels of these cytokines.


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IL-1b

TNFa mRNA % WT

SPE7

SPE7

MCP-1

IL-6 mRNA % WT

SPE7

SPE7

FIG. 4. Defective sensitization in the absence of IgE is associated with reduced gene expression. Cytokine transcripts were measured by RT-PCR 1 h after oxazolone application in wild-type (WT) and IgE-/- mice and in IgE-/- mice 18 h after i.v. injection of monoclonal IgE antibody, SPE-7.

Taken together, these findings suggested that the cytokine milieu of IgE-/- skin is not optimally supportive of immune sensitization to haptens. Furthermore, the data indicated that IgE, even in the absence of known specific antigen, could enhance hapten-induced cutaneous levels of a number of mRNAs for several mediators that are known to influence dendritic cell function and immune sensitization. These studies have identified a novel function for IgE antibodies in regulating cutaneous immune sensitization to haptens. Using passive reconstitution with IgE, cellular adoptive transfer and analysis of dendritic cell numbers in the epidermis, we have established that optimal immune sensitization to chemical haptens requires, at the time of antigen exposure, the presence of IgE. In our model, IgE clearly is not required at the elicitation phase of contact sensitivity, as shown by the normal ear swelling responses that are generated in IgE-/- mice that have been adoptively immunized with cells derived from oxazolone-sensitized wild-type mice. The data strongly support the hypothesis that the effects of IgE during hapten sensitization involve FceRI, mast cells and mast cell-derived products and that IgE can contribute to optimal sensitization for contact sensitivity by influencing dendritic cell mobilization (Fig. 5). While our experiments evaluated levels of mRNA for cytokines IL1b, IL6 and TNFa and a chemokine (MCP-1) that can influence dendritic cell

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FIG. 5. Effects of IgE and mast cell on cutaneous immune sensitization. IgE antibodies, interacting with mast cells via FceRI, induce increased IgE receptor expression and also prime mast cells for the production of TNFa, IL6, IL1 and MCP-1, and probably other cytokines, following skin exposure to contact sensitizers. These cytokines collaborate to drive dendritic cell activation, migration to regional lymphoid tissue and effective immune sensitization of antigenspecific T cells.

migration or maturation, mast cell histamine represents another mediator that can influence dendritic cell migration from the epidermis, at least when the mast cells have been activated by IgE and specific antigen ( Jawdat et al 2004). One concern that has been raised regarding studies of ‘monomeric’ IgE on mast cell functions in vitro has been the possibility of artifactual induction of responses by ‘non-physiological’ IgE aggregation. Our data clearly establish that contact sensitivity responses can be impaired in IgE-/- mice in comparison to the responses that are expressed in the corresponding wild-type mice, establishing an apparently antigen-independent role for IgE in an in vivo system that is free of exogenous physical or biochemical factors that might induce non-physiological aggregation of IgE. As mentioned in the introduction, the mechanism(s) whereby IgE might activate mast cells in the absence of known antigen has/have not yet been elucidated. Engagement of FceRI by its ligand, monomeric IgE, may provide a signal in and


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of itself. Alternatively, receptor-bound IgE antibodies might have some tendency to associate in the absence of antigen, leading to FceRI aggregation and signalling. Finally, it has been suggested that molecules other than nominal antigen could interact with some IgE antibodies. James and colleagues observed that a single IgE antibody, SPE-7, can exist in at least two isomers, one of which binds to DNP and another which binds to an unrelated protein antigen ( James et al 2003). SPE-7 has relatively potent mast cell simulating properties in culture (Kitaura et al 2003) and, in our hands, SPE-7 was somewhat more effective than the other IgE antibodies studied in restoring both ear swelling responses and skin cytokine gene expression. It is possible that such ‘antigenic promiscuity’ might underlie the interesting effects of SPE-7 observed by us and others. However, attributing reconstitution of contact sensitivity responses in IgE-/- mice by each of the four different IgE monoclonal antibodies (which have hapten and protein specificities unrelated to each other and to oxazolone) to such alternative antigen binding would require that they each have an alternative isoform capable of binding either oxazolone or an unrelated endogenous antigen. Although this might seem far-fetched, given the lack of precedent for such a phenomenon among other immunoglobulin isotypes, it is possible that some low-affinity ‘alternative’ antibody-antigen interactions may become evident because of the intense biological amplification potential of the IgE/FceRI/mast cell system. Summary In conclusion, we have defined a previously unknown function of IgE antibodies, namely, facilitating the sensitization phase of contact sensitivity responses. Remarkably, although this IgE effect is mediated via FceRI and mast cells, it does not require exposure to antigens for which the IgE has known specificity. We hypothesize that a potential mechanism whereby IgE can support optimal immune sensitization involves the maintenance of a cutaneous cytokine milieu favourable to dendritic cell activation and T cell sensitization (Fig. 5). Acknowledgements HCO is supported by NIH/NIAID 1-R01-AI054471 and SJG and MT are supported by NIH R01-CA72074, R37-AI23990 and P01-HL67674 (Project 2).

References Asai K, Kitaura J, Kawakami Y et al 2001 Regulation of mast cell survival by IgE. Immunity 14:791–800 Beck LA, Marcotte GV, MacGlashan D, Togias A, Saini S 2004 Omalizumab-induced reductions in mast cell FceRI expression and function. J Allergy Clin Immunol 114:527–530

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Bryce PJ, Miller ML, Miyajima I et al 2004 Immune sensitization in the skin is enhanced by antigen-independent effects of IgE. Immunity 20:381–392 James LC, Roversi P, Tawfik DS 2003 Antibody multispecificity mediated by conformational diversity. Science 299:1362–1367 Jawdat DM, Albert EJ, Rowden G, Haidl ID, Marshall JS 2004 IgE-mediated mast cell activation induces Langerhans cell migration in vivo. J Immunol 173:5275–5282 Kalesnikoff J, Huber M, Lam V et al 2001 Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 14:801–811 Kisselgof AB, Oettgen HC 1998 The expression of murine B cell CD23, in vivo, is regulated by its ligand, IgE. Int Immunol 10:1377–1384 Kitaura J, Song J, Tsai M et al 2003 Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI. Proc Natl Acad Sci U S A 100:12911–12916 Oettgen HC, Martin TR, Wynshaw-Boris A et al 1994 Active anaphylaxis in IgE-deficient mice. Nature 370:367–370 Pandey V, Mihara S, Fensome-Green A, Bolsover S, Cockcroft S 2004 Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2+, degranulation, and membrane ruffling in the cultured rat basophilic leukemia-2H3 mast cell line. J Immunol 172:4048–4058 Yamaguchi M, Lantz CS, Oettgen HC et al 1997 IgE enhances mouse mast cell Fc(epsilon)RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgEdependent reactions. J Exp Med 185:663–672 Yamaguchi M, Sayama K, Yano K et al 1999 IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord bloodderived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release. J Immunol 162:5455–5465

DISCUSSION Metcalfe: What do you anticipate you would see if you were to select people with different levels of IgE and then look at mast cells in the skin? If you selected a person with a very high IgE level, how would the mast cells in the skin compare with someone who had half this IgE level or very low IgE levels? Would there be different levels of priming? Oettgen: That’s a great question and I think it would be worth looking at in humans. In mice there is an interesting paradox with regard to the hypothesis I created with my introduction, that although there is a clear difference in cytokine levels between the skin of IgE-deficient mice and wild-type mice (which have physiologically low IgE levels), there is no further increase in contact sensitivity responses or in cytokine level as we go into an allergic state. If we get the IgE levels to go even higher, by injection we can’t show a further shift either in the contact sensitivity response or in dermal cytokine levels. Metcalfe: In the human, however, your speculation is confounded because human dendritic cells have IgE. Metzger: In one sense, we could rephrase the issue that you are raising in terms of antigen-independent sensitization: is it independent of aggregation of the receptor? Your cartoon suggests that it is.


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Oettgen: The cartoon does, but the data don’t. Metzger: This is what I was wondering about. To some extent, what I am asking is as follows. When you do your reconstitution with specific hybridoma IgEs, you can mimic the effect of the wild-type mice. This suggests that at least some of the data in the past, where the suggestion was that it is only specific hybridomas that tend to aggregate either spontaneously or because of some unknown antigen in the serum in which the cells are grown, may not be correct. On the one hand the fact that the wild-type mice, which presumably have heterogeneous IgE, show the same effect as the hybridoma, is a control. But on the other hand, we don’t know how heterogeneous the IgE in the wild-type mice is. Might it be worthwhile doing an experiment in which, instead of adding one hybridoma IgE, you add a mixture or you titrate the different IgEs on the basis of their aggregating ability or stimulatory activity? If you titrate the IgEs so that the one that is the most stimulatory, such as SPE-7, just gives you a sensitization, and then you use equivalent amounts of a hybridoma that doesn’t stimulate, this might provide some useful information. Oettgen: I don’t think we can distinguish from our data whether the signalling that is induced by IgE involves aggregation or not. It may or may not. I think the strength of the study is that wild-type mice behave differently from IgE-deficient mice. If there is some aggregation, this might be something that is physiological or happens under normal circumstances. Your idea about looking at the different antibodies is a good one. We have looked at a number, but as you implied we only looked at a single dose. The single dose–response experiment that I showed for reconstitution was done with the SPE-7 antibody. Each of those lines represents at least 10 mice. We haven’t done it with all the other clones to see whether there is a dose–response relationship in terms of reconstitution of contact sensitivity that parallels the cytokinergic capacity of the antibodies as Toshi Kawakami defined them in his culture systems. Metzger: Would it be interesting to use an anti-receptor antibody to see whether this will mimic the presence of IgE? Oettgen: We are trying to do those experiments. Jean Marshall has data on this. Did you use anti-IgEs? Marshall: We just used IgE and antigen. Rivera: Do you know that the mast cells in these IgE-deficient mice are expressing the same number of receptors as wild-type cells? The fact that the cytokine milieu is different in those mice might also imply that the priming of these mast cells is different. The in vitro data that Toshi Kawakami and Gerry Krystal have generated suggest that there is a dose-dependent effect in terms of the IgE concentrations required for mast cell activation. Have you used supraphysiological concentrations of IgE? Oettgen: The only data that we have about in vivo receptor numbers were data that were generated by Steve Galli’s group. Peritoneal mast cells in IgE-deficient mice had decreased FceRI.

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Galli: The levels were decreased by approximately 80% versus those in the corresponding wild type mice (Yamaguchi et al 1997). Oettgen: We could reconstitute this by i.v. injection of IgE, so there is a diminished FceRI density. This is interesting because the B cell levels of CD23 are also markedly diminished and will come up after IgE injection. The IgE ligand is positively regulating both of its receptors in this system. In terms of supraphysiological levels of IgE, I don’t think we showed that this got the levels of high-affinity IgE receptor any higher than physiological levels. Galli: As I remember those experiments, we only tested a single condition of IgE transfer in vivo because of the expense of the mice. However, in experiments performed in vitro, it was possible to drive numbers of FceRI on mast cells to very high levels by subjecting the cells to high concentrations of IgE. The idea that the ligand, IgE, might regulate the level of surface expression of its receptor, FceRI, is not a new one, as there were at least two pieces of evidence in the literature, based on studies of rat basophilic leukaemia (RBL) cells, indicating that this could occur (Furuichi et al 1985, Quarto et al 1985). The key new findings which we reported were that this also occurs in normal mast cells, in vivo as well as in vitro, and that the ability of IgE to increase levels of surface expression of the FceRI has biologically significant consequences, in that this can enhance the cells’ IgE-dependent effector function. Another complexity should be kept in mind when considering the potential roles of mast cells in contact hypersensitivity, namely, some examples of contact hypersensitivity appear to be expressed independently of mast cells. You may recall that Phil Askenase’s group was the first to report that, in certain examples of contact hypersensitivity, the expression of the response was diminished in the absence of mast cells (Askenase et al 1983). In contrast, my group (Galli & Hammel 1984, Mekori & Galli 1985, Mekori et al 1985, Mekori et al 1987), John Schrader’s group (Thomas & Schrader 1983) and T.-Y. Ha et al (Ha et al 1986), published other examples of contact hypersensitivity responses in which there were no defects apparent when the responses were elicited in the absence of mast cells. If you examine these reports in detail, you will see that there were differences in the contact hypersensitivity protocols that were being tested in these different laboratories, and, in some cases, different assays were used to measure the responses. Given the findings in the experiments that we conducted with Hans Oettgen (Bryce et al 2004), and which he has just described, we re-examined this issue. We have confirmed that, with certain combinations of haptens and vehicles, no defect in the tissue swelling associated with the expression of the contact hypersensitivity responses is detectable in the absence of mast cells. Thus, mast cells can be shown to make an important contribution to the expression of certain examples of contact hypersensitivity responses, but not others. I think that it will be of interest to understand why this is so. Metcalfe: Which haptens work versus those that do not?


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Galli: As Hans Oettgen showed, when dinitrofluorobenzene (DNFB) was administered in ethanol as the vehicle, the contact hypersensitivity response was partially mast cell-dependent. However, when we tested DNFB in acetone, there was no demonstrable defect in the tissue swelling associated with the response in the absence of mast cells. Oettgen: In general, if you look at all these studies, then acetone seems to be something that gets rid of the mast cell requirement. Is that your impression? Galli: It seems so, although the mechanism for that has not been elucidated. Austen: Steve’s comment prompts me to ask this. Your analysis of the site of the IgE requirement was in the sensitization and not the elicitation step. If we focus on that, it is conceivable that different haptens might give a different readout. It is not just a matter of sensitization; the elicitation at a very different dose is with the effector phase of the entire response. This reminds me of studies looking at the migration of dendritic cells (DCs) to the regional nodes. They do not necessarily go on to the challenge step, but look at what regulates the movement of cells from the periphery to the nodes. Marshall: In the context of this study of the mast cell interaction with the Langerhans cell, there is frequently an assumption that the Langerhans cell in particular is this one stage of cell. Yet there may be a number of maturational stages in terms of how the Langerhans cell takes up residence and becomes responsive. You have potentially only 20% of the cells responding at a given time. In the context of your cartoon you show oxazolone interacting with the mast cell, which then interacts with a dendritic cell. Though I am a big proponent of mast cell-dependent dendritic migration, another potential model is that the mast cell provides a maturational signal through these changes in cytokine production which then enables the Langerhans cell to respond to a contact sensitizing agent. Have you thought about this? Oettgen: I have thought about it, but I haven’t addressed this. This is one possible hypothesis: the mast cell is primed and then irritated by either the vehicle or the oxazolone-produced factors, which then act on Langerhans cells in the skin. But it is also conceivable that the mast cell is consistently producing factors which affect Langerhans cell phenotype in the skin in such a way as to alter their response when they come in contact with the hapten. We haven’t started to look at this, but it is a good idea. Marshall: It is also more consistent with the diversity in terms of the sensitizing agent combinations that work. Oettgen: As you pointed out in your initial question, the limited number of dendritic cells that leave the skin is consistently only about 20%. Perhaps they were different to start with in terms of their phenotype, rather than being induced at the time of sensitization to be activated. Pecht: Coming back to the chemistry, with oxazolone you are apparently producing its (hapten-) conjugated proteins. These, in turn, provide the clustering

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agents for the IgEs. Hence the question of monomeric IgE becomes irrelevant. As soon as you are exposing the animal to the so-called monovalent haptens which are reactive chemicals these will react with encountered proteins producing polyvalent antigens and eliminate the issue of monomeric IgE being the reactive species. Oettgen: At the sensitizing phase, which is when we see the difference in phenotype, even though there are poly-oxazolonated peptides, there shouldn’t a priori be IgE that interacts with them as an antigen. Pecht: You started off by showing us results which clearly show wide crossreactivity. For sure, if you are adding the specific hybridoma it is occurring, and I suspect that it will also happen with other hybridomas. Oettgen: Your point is well taken. I tried to avoid using that term, ‘monomer’. Pecht: This is a significant issue. Namely, is it the monomeric IgE that is doing the job, or is it a regular antigen–IgE reaction that takes place? Oettgen: As you point out, I don’t think this can be distinguished. MacGlashan: I want to follow up on Henry Metzger’s question. One of the interesting experiments done with SPE-7 was to include monovalent hapten and show that all the capabilities of SPE-7 were ablated. I don’t know whether this can be done in vivo, but an experiment you could try is to use SPE-7 purposely and put in a load of monovalent antigen, to see whether you can knock out that response. Oettgen: That is a good idea. Pecht: My suggestion would be to use a chemically inert reagent that is not capable of reacting with protein components within the animal. Use for example, dinitrobenzene rather than a reactive derivative of it. Oettgen: Is that going to be contact sensitizing? Pecht: No, it probably is not for the very same reason that it is relatively inert but one should try to employ the least reactive chemical for such a purpose. Razin: I looked in the literature and found that some reports show that just monomeric IgE triggers mast cells to degranulate. Some other reports say that this is not the case. Toshi could comment on this: what is the situation? Kawakami: I don’t think it is important whether IgE aggregates in solution. However, aggregation of receptor takes place with monomeric IgE or IgE plus antigen, at least in vitro. In his case, the presence of IgE leads to the release of several cytokines and chemokines. This is the most important aspect, although I agree that the experiment with dinitrophenyl (DNP) lysine together with SPE-7 would be nice to do. Razin: What about degranulation and histamine release? Kawakami: We have seen histamine release with SPE-7 IgE for sure. Koyasu: I have a question about the dendritic cell movement. You show that various factors can be affected by the absence of IgE, including IL1, IL6 and MCP1. I am surprised that TNFa didn’t show any effect on the movement of dendritic


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cells in your experiment, because we know that it can affect the dendritic cell maturation. Does this mean that TNFa is already provided by epidermis in your experiment, and therefore it is not the limiting factor? Oettgen: With TNF, we have only tried one internal dose. It did give a hint of some reconstitution. Clearly there was a lower level of TNF in IgE-deficient animals. All the cytokines could be coming from other cells. One thing our study doesn’t do in terms of the PCR analysis is to establish that the defect in the cytokine levels in the skin is really at the level of the mast cell. Koyasu: This leads to a different question. When you used different types of solvent you obtained different results. One is dependent on mast cells and one is independent. Another experiment would be to take dermal sheet from the Ce knockout and use different types of stimuli, to see whether there is migration of DCs from the dermal sheet even in the absence of IgE. Oettgen: I think that is a good idea. It would help establish whether the difference that exists that is being reported in terms of contact sensitivity responses depending on sensitizer, might be related to the efficiency with which dendritic cell emigration is promoted. Koyasu: Can you do the experiment using the neutralizing antibody to these cytokines with the dermal sheet, for example, to block the migration of the dendritic cells? Oettgen: We haven’t done this. Ono: I noticed that in your cytokine add-back experiments, the kinetics of rescue were very different. Usually they resolved very quickly when you added certain cytokines; in other cases it was prolonged. What do you make of this? Oettgen: You are probably alluding to the fact that the TNF response dropped off in animals that received intradermal TNF prior to sensitization. They developed a contact sensitivity response that fell off very quickly. Ono: I am referring to the experiment in which you injected each of these different mast cell derived chemokines and looked at the ear swelling response. Oettgen: IL6 had a robust and sustained response. The rapidity with which the response waned was also related to its magnitude. The more robust reconstitutions tended to stay up longer and the less robustly reconstituted responses dropped off more quickly. Ono: It had nothing to do with the nature of the inflammatory infiltrate then. Oettgen: We didn’t look. Metzger: I’d like to go back to the beginning of Hans’ paper where he mentioned the patient with multiple allergies. There is the implication there that the spread of allergies to a variety of antigens may not simply be due to the fact that these people tend to produce IgEs against things that they shouldn’t, but that in fact the initial allergies are promoting the subsequent sensitivity or increase the chance of getting other allergies. I’d like to ask the clinicians whether there is a therapeutic implica-

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tion here that if you would treat the initial allergies vigorously, it might prevent this kind of spread. Metcalfe: There is modest speculation on asthma in terms of genesis with a known antigen and epitope spreading, where the IgE then recognizes a human component. The idea is that the prolonged phase of asthma may have an immune component. If you follow this logic, the earlier you could control the IgE response to a given antigen, the less likely there would be epitope spreading. This would suggest a strong rationale for early intervention. It doesn’t have to be by some kind of immunotherapy. It could be, as the paediatricians are advocating, by early use of steroids to dampen the inflammatory response. Metzger: His mechanism would not be due to epitope spreading, but rather sensitization. The epitope spreading has been the traditional explanation. It seems that these experiments suggest there may be another mechanism by which one gets spreading, due to enhanced sensitization of some of the cells that lead to the effector cells. Metcalfe: Focusing on effector cells, I would think would lead to a similar conclusion; that the early treatment of inflammation, say with steroids, would have a beneficial effect on early sensitization. Have you examined how long it takes to sensitize from when the sensitizing agent is applied? This would provide some insight into the processes. When you administer IgE to an IgE-deficient mouse, have you studied how long it takes for the sensitizing agent to be effective? Rao: Or even more simply, if you made mast cells from the bone marrow of these mice and added irrelevant IgE for a certain time, and came back with a specific antigen–IgE complexes, would you get an increase in the level of the response? Oettgen: We have only looked at this under one condition. IgE half-life in the mouse is so short we have tried injecting the IgE very shortly before the sensitization. We haven’t tried more prolonged experiments. It was the day before we did the experiment. Metcalfe: Does the IgE have to be added systemically? There is trafficking going on. I am curious as to whether the cells in the skin that are already there can simply be turned on by adding IgE locally, or whether or not there is a more general phenomenon. Oettgen: We have only done it that way. We have avoided the intradermal injection for fear that injection would elicit a cytokine response in terms of injury that would obscure the result. We should try it. Rao: We are looking at the mechanism of some of these cytokine expression events in mast cells. If IgE-deficient mice are giving you less of this response and you need IgE sensitization, can you recapitulate this in vitro in mast cell culture, and look at what the mechanism of sensitization is? Have you somehow primed at a nuclear level? Have you made pre-mRNA that is spliced more efficiently?


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Oettgen: If we grow bone marrow cells from our IgE-deficient mice it would take weeks, which puts us off a bit. You are suggesting that something happened that had a permanent genetic effect on the mast cells? Rao: It could be accumulation of unspliced message, and then when you put on the Fc receptor–antigen complexes you get rapid splicing. Or it could be a protein that is made in an intracellular pool that traffics acutely to where it is needed. Oettgen: But you could envisage something that permanently affects the mast cell gene expression. Rao: It could be something that takes three days to develop and then three months to go away. Stevens: The IgE-binding factor that Dr Liu’s group identified in the 1980s (Liu et al 1985) is now known as galectin 3 (GenBank Locus ID 3958). Galectin 3 is a galactose-binding lectin. Thus, in your in vivo priming of mast cells, it is possible that you are inducing the expression of galectin 3 or a related lectin that binds to IgE or its receptor thereby affecting FceRI signalling pathways? Oettgen: We haven’t looked at galectin. Rivera: I have a question for the clinicians in this audience. What is the relationship between atopy and the contact sensitivity? Oettgen: There isn’t a clear relationship. Some people with atopic eczema develop contact sensitivity, but there doesn’t seem to be a greater susceptibility. Metcalfe: Contact sensitivity in humans is as complex as it is in the mouse. There are some forms where there is an IgE phenomenon early and others where there isn’t. As far as I know, people with mastocytosis don’t have any change in contact sensitivity. I am sure that there are multiple mechanisms. Oettgen: People with atopic dermatitis are clearly able to become sensitized to additional antigens through the skin. Marshall: In the studies that we have done recently we have shown that IgEmediated mast cell activation which leads to Langerhans cell migration is H2 dependent. Have you looked at this at all? Oettgen: No. You used an H2 histamine receptor blocker to show this. We haven’t tried to use histamine receptor blockers in this system. Walls: Your focus has been on contact sensitivity. What conditions other than contact sensitivity in the skin might your findings be relevant to? For example, T cell-mediated events in the synovium could involve similar processes. Oettgen: Our particular interest is in the lung, so we are trying to look at respiratory mucosal responses to antigen. We have tried to approach this by using contact sensitizers in the lung. It is a mouse version of occupational asthma in humans. So far we have had a lot of difficulty reproducing the phenomenon that we observe in the skin in the lung. This may relate to the irritant effect of the vehicles or the mast cell distribution in the airway. In the mice there aren’t many mast cells in the

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lower airway. We have started in lung but we also want to look at different kinds of immune responses, for instance to protein antigens delivered via various routes. Walls: So you see this as a function of the mast cell or dendritic cell heterogeneity? Austen: We probably shouldn’t mix contact sensitivity with allergen challenge in the pulmonary models. In the pulmonary model a mast cell product, PGD2, is an inhibitor of the dendritic cell movement. This has not yet been studied in the contact sensitivity model. MacDonald: Many of you here will recognize that I put forward a concept of IgE plus and minus in humans. This definition was based on a conversation I had with Kimi Isizaka. When I heard Toshi talk and read Gerry Krystal’s paper (Kalesnikoff et al 2001) I felt thank goodness we have a mechanism. I want to say a couple of things about human basophils. First, unfortunately SPE-7 does not bind human basophils. There are some mouse IgEs that do bind, but SPE-7, being commercially available and rather cheap is not one of them. Second, I have purified IgE from HRF responders, who by definition are IgE plus responders, and compared this with IgE purified from IgE non-responders. Neither of these produce ERK phosphorylation by themselves. I am left with the question of some sub level aggregation in the human system rather than a monomeric concept. Rivera: Was the concentration of IgE used for your experiments equivalent to what has been used in previous studies, which was as much as milligram amounts of IgE? This is a key issue, at least in the in vitro studies. MacDonald: I believe that in the mouse system it has been up to 10 mg/ml sensitization. We didn’t get to 10, but we did get to 1 mg/ml. Rivera: The SPE clone-derived IgE seems to activate in the microgram range, but other IgE clones require concentrations in the milligram range. Kawakami: We have evidence that SPE-7 IgE can synergize with mouse histamine releasing factor on mouse mast cells in terms of cytokine production and also survival enhancement. Only SPE-7 IgE can do this, and not so-called poorly cytokinergic IgEs. Galli: I want to make two technical comments about experiments investigating interactions between mast cells and dendritic cells. One of them has already been alluded to: one has to consider many aspects of how the mast cell could influence dendritic cell biology, from migration, to influence on phenotype/maturation, to functional effects. While one can readily measure egress or movement of dendritic cells, it may require the use of special methods to investigate effects of mast cells on these other aspects of dendritic cell biology. The second point is that while data concerning the effects of mast cells on dendritic cells can be very informative, ideally one would also attempt to quantify the extent of expression of the final acquired immune response which is elicited in the animal. The dendritic cell can influence such aspects of acquired immune responses as whether or not they occur, their intensity, and so forth. So, there really are two important questions. First, does


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the mast cell influence dendritic cell biology? Second, are such effects of mast cells on dendritic cells significant in the development of the final immune response? Depending on the experiment, it is possible that sometimes the answer to the first will be yes but the second will be no. Ono: In your IgE-deficient mouse models, you can really overshoot the concentration of IgE that you put into the animal. You made some sort of a comment that this really didn’t affect mast cell function as opposed to other types of activity. Is that correct? Oettgen: The thing we have looked at directly is the contact sensitivity response after we overshoot. By massively overshooting physiological IgE levels you can never get above wild-type contact sensitivity responses. The way we have tried to get at this issue a little better is to make a wild-type hypersensitivity response less robust by cutting back on the sensitizing dose of hapten, to see whether we could get below a threshold. We found that we could go down logs on the sensitizing dose and still sensitize very effectively. When we got to threshold levels the system became too stochastic for analysis. Ono: What is the magnitude of excess IgE over the threshold in your system? Oettgen: We were using IgE reconstituting doses that gave a plasma level at least a log or more higher than physiological levels. MacGlashan: I want to follow something up that Rick said about CD23 being involved in this. I don’t know the biology of how CD23 could work towards priming a mast cell, but the fact is that there is something stabilizing this interaction as well, preventing the release of soluble CD23. It could be a very indirect effect on the mast cell mediated by IgE, I suppose. Oettgen: The IgE-deficient mice might have greatly elevated levels of soluble CD23. MacGlashan: Your high affinity IgE receptor deficiency would speak to that. Brown: Is there anything known about the ability of the reagents used in sensitivity models to directly activate or sensitize the mast cells? Are they inducing any signalling events themselves? Oettgen: I am not aware of published knowledge on this. We have tried to do crude experiments. Brown: There is some evidence that allergens that are serine proteases can directly activate eosinophils (Reed & Kita 2004). We don’t think about the potential contribution of these effects. Metzger: He has shown that receptor deficiency blocks the response. Brown: Yes, but perhaps both components are needed for the optimal response. Austen: Has this phenomenon of IgE function without obvious cross-linking been observed in human mast cells or basophils? Bradding: We have looked in human lung mast cells that we have kept in culture for several weeks to remove endogenous IgE. A number of cells die in the first

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week of culture and then we get proliferation. When we add IgE to those cells we do see responses. We see degranulation, leukotriene production and IL8 release (Cruse et al 2005). Galli: Using umbilical cord blood-derived mast cells, we have found that a preparation of commercially available human myeloma IgE will enhance the survival of human cord blood-derived mast cells upon withdrawal of SCF. Again, we are dealing with IgE in the 1–10 mg range. Such treatment will also induce the release of certain chemokines, but not histamine. The effect on chemokine release is a consistent observation with multiple batches of umbilical cord blood-derived mast cells, each of them being genetically unique. It appears that whatever this phenomenon is, it is not restricted only to mice. Austen: I want to be clear on the assays. There are others who haven’t been able to reproduce this, so I want to tell them at least what assay they should look at. Can you say more about the survival assay and the factors that you mentioned? Galli: The assays are quite similar to those used with mouse mast cells. There is a period during which one incubates the human mast cells with SCF. We usually use them about 10–16 weeks after starting the culture of umbilical cord blood cells. Then, SCF is either maintained or withdrawn. Once SCF has been withdrawn, IgE is added in concentrations of 0.1, 1 and 10 mg/ml. Generally, the most striking effect on survival, which is assayed 48–72 h later, is with the 10 mg/ml dose. Austen: What is the survival assay? Galli: We double stain with FITC-conjugated annexin V and propidium iodide and, in some experiments, confirm the results by counting the number of mast cells that survive. Essentially, the assay is quite similar to that performed by Toshi Kawakami with mouse mast cells. We also searched for histamine release, and saw none. Nor did we detect IgE-induced release of lipid mediators. But we have detected the IgE-dependent release of a couple of chemokines. One problem is that a large panel of monoclonal IgEs, which is available in the mouse, is simply not available in the human. MacGlashan: We have tried several IgEs. In general, we cannot see any response or signalling. There is no indication that you can improve survival, although this situation is so different from the mast cells grown in culture I am not sure it means much. PS myeloma is the one IgE that occasionally we have seen a bit of signalling with at very high concentrations. The problem is that we haven’t really processed that myeloma for the elimination of aggregates. The fact that it is sporadic led us not to pursue this. Also, when we did some work with Hannah Gould she would say that PS myeloma is odd in a number of biophysical parameters. Without any further study, I can’t really speculate. Razin: It could be that all the receptors are already occupied by IgE, because you purified the basophils from the blood.


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MacGlashan: When we were really trying to induce something we would remove the endogenous IgE by a brief mild acidification of the buffer. Razin: What was the cell viable assay? You remove the KIT ligand, add the IgE and prevent apoptosis. Galli: It reduces the rate of apoptosis. All the cells will die eventually. Metcalfe: Don, I’m assuming you haven’t tried to do this experiment in cultured human basophils. MacGlashan: These are not cultured basophils, or grown from progenitors. Metcalfe: In normal physiology, both human mast cells and basophils see IgE as they differentiate. I am curious as to how you can replicate any effect only due to the addition of more IgE in a physiological situation. I can understand why if a mast cell or basophil has never seen IgE throughout its development that you could observe some rather profound effects if you then added IgE and measured carefully. I wonder if there is any way experimentally to see if this has any relevance to responsiveness of a mast cell or basophil that has been constantly in the presence of IgE during its development phase so that IgE receptors placed on the surface are immediately exposed to IgE. Cockcroft: We can sensitize the RBL cells with IgE overnight at low concentrations. The next day we can come along and stimulate with monomeric IgE, and the cells remain responsive. Razin: What do you mean by ‘respond’? Cockcroft: That we can stimulate to degranulate with just IgE. These cells have been sensitised overnight. I can put in 200 ng IgE overnight. We have to titrate down the concentration of SPE-7. The fact that the cells have seen IgE, doesn’t necessarily mean that they won’t respond to IgE again. Metcalfe: In the physiological situation, mast cells or basophils are never in the absence of IgE. All through development they have seen it. There may be effects on development with and without IgE, but in the physiological situation it is hard to understand how simple addition of more IgE can be relevant. Rao: In almost every signalling system that people have worked on, the basal level of signalling is important for setting the subsequent response. Tada Taniguchi has best demonstrated this for the interferon response (Taniguchi & Takaoka 2001): if he knocks out basal interferon signalling by taking out receptors or relevant transcription factors he gets a much attenuated response. Galli: As Hans Oettgen reported, under basal conditions his IgE knockout mice exhibit no reductions in numbers of mast cells in the several anatomical sites which he examined. So, it appears that in normal mice with normal levels of mast cells, and in the absence of an inflammatory or immune response, there is no detectable contribution of IgE to mast cell numbers, at least in the anatomical sites which were analysed. However, Hans and his colleagues showed that during infection with

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Trichinella spiralis, the numbers of mast cells in the spleen were significantly elevated in the wild type mice as compared to the IgE-/- mice (Gurish et al 2004). The experiment that we did with Toshi was to take either IgE-producing or IgG2bproducing hybridomas, inject them into the peritoneal cavity of normal mice, and then two weeks later count mast cell populations in various organs. This showed a significant positive correlation between concentrations of IgE in the blood and numbers of mast cells in the gastrointestinal tract (Kitaura et al 2003). Thus, at high concentrations in vivo, IgE seems to be able to influence the size of certain populations of mast cells. However, currently available evidence indicates that, in the mouse, this IgE-dependent effect on mast cell numbers is quantitatively small compared with that attributable to SCF/KIT signalling or even to IL3. Metcalfe: Does anti-IgE ever lower the IgE level low enough that re-establishing an IgE level or having it come back to normal would have an effect? MacGlashan: I would think that they would get low enough. In the original phase I trial free IgE levels were around 5 ng/ml. This is very low. It is also low enough to reduce the cell expression of the receptor to a steady state. About 10000 receptors per cell is where the cell will naturally sit even if there is no IgE around, and in those studies, that’s where it went. Metcalfe: That is free IgE. My assumption would be that there is a significant reservoir of bound IgE. MacGlashan: With that approach you certainly can’t eliminate the IgE completely. Razin: Hans Oettgen in your experiment what proportion of the injected IgE went to mast cells? Oettgen: We didn’t test this. Razin: This is important. Galli: The effect was not observed in animals that lack the g chain common to FceRI and Fcg RIII. Razin: Yes, this is a good indication, but what I still haven’t seen is whether what you injected is on the mast cell surface. Austen: Some will be on the mast cells because that’s the highest affinity place to catch it. Oettgen: We know the injected IgE is cleared very quickly, but we can’t distinguish from this destruction versus binding to mast cells, as you say. We haven’t labelled mast cells with anti-IgE. Razin: It is easy to do. You can label IgE and follow it. Metcalfe: If you take mast cells from IgE-negative mice and grow them up in the presence of IgE, is it possible to inject these IgE-positive mast cells from the same mouse back into the skin and then sensitize? This would help differentiate effects of CD23, effects on dendritic cells and so on. Oettgen: There is a precedent for doing intradermal reconstitution of mast cells, but we haven’t done what you suggest.


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Galli: The first time we used mast cell-engrafted W/W v mice for functional studies of mast cells, in addition to doing long-term mast cell engraftment we injected in vitro-derived cloned mast cells and then tested their function the next day (Wershil et al 1987). This tells us that such mast cells can function in vivo within 24 h of being removed from culture. Oettgen: Do you think they would reconstitute a mast cell-sufficient recipient? Galli: Professor Kitamura has already shown that the higher the numbers of mast cells that are already present in the skin, the more difficult it is to introduce more of them. References Askenase PW, Van Loveren H, Kraeuter-Kops S et al 1983 Defective elicitation of delayedtype hypersensitivity in W/W v and Sl/Sl d mast cell-deficient mice. J Immunol 131:2687– 2694 Bryce PJ, Miller ML, Miyajima I, Tsai M, Galli SJ, Oettgen HC 2004 Immune sensitization in the skin is enhanced by antigen-independent effects of IgE. Immunity 20:381–392 Cruse G, Kaur D, Yang W, Duffy SM, Brightling CE, Bradding P 2005 Activation of human lung mast cells by monomeric IgE. Eur Respir J 25:858–863 Furuichi K, Rivera J, Isersky C 1985 The receptor for immunoglobulin E on rat basophilic leukemia cells: Effect of ligand binding on receptor expression. Proc Natl Acad Sci USA 82:1522–1525 Galli SJ, Hammel I 1984 Unequivocal delayed hypersensitivity in mast cell-deficient and beige mice. Science 226:710–713 Ha T-Y, Reed ND, Crowle PK 1986 Immune response potential of mast cell-deficient W/W v mice. Int Arch Allergy Appl Immunol 80:85–94 Kalesnikoff J, Huber M, Lam V et al 2001 Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 14:801–811 Liu FT, Albrandt K, Mendel E, Kulczycki A Jr, Orida NK 1985 Identification of an IgE-binding protein by molecular cloning. Proc Natl Acad Sci USA 82:4100–4104 Mekori YA, Galli SJ 1985 Undiminished immunologic tolerance to contact sensitivity in mast celldeficient W/W v and Sl/Sl d mice. J Immunol 135:879–885 Mekori YA, Weitzman GL, Galli SJ 1985 Reevaluation of reserpine-induced suppression of contact sensitivity. Evidence that reserpine interferes with T lymphocyte function independently of an effect on mast cells. J Exp Med 162:1935–1953 Mekori YA, Chang JC, Wershil BK, Galli SJ 1987 Studies of the role of mast cells in contact sensitivity responses. Passive transfer of the reaction into mast cell-deficient mice locally reconstituted with cultured mast cells: effect of reserpine on transfer of the reaction with DNP-specific cloned T cells. Cell Immunol 109:39–52 Quarto R, Kinet J-P, Metzger H 1985 Coordinate synthesis and degradation of the alpha-, beta- and gamma-subunits of the receptor for immunoglobulin E. Mol Immunol 22:1045– 1051 Reed CE, Kita H 2004 The role of protease activation of inflammation in allergic respiratory diseases. J Allergy Clin Immunol 114:997–1008 Taniguchi T, Takaoka A 2001 A weak signal for strong responses: interferon-alpha/beta revisited. Nat Rev Mol Cell Biol 2:378–386 Thomas WR, Schrader JW 1983 Delayed hypersensitivity in mast-cell-deficient mice. J Immunol 130:2565–2567

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Wershil BK, Mekori YA, Murakami T, Galli SJ 1987 125I-fibrin deposition in IgE-dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells. J Immunol 139:2605–2614 Yamaguchi M, Lantz CS, Oettgen HC et al 1997 IgE enhances mouse mast cell Fc(epsilon)RI expression in vitro and in vivo: Evidence for a novel amplification mechanism in IgEdependent reactions. J Exp Med 185:663–672

The role of Src family kinases in mast cell effector function Yasuko Furumoto, Gregorio Gomez, Claudia Gonzalez-Espinosa*, Martina Kovarova, Sandra Odom, John J. Ryan† and Juan Rivera1 Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 208921820, *CINVESTAV Zona Sur, Mexico D.F., CP14330, and †Virginia Commonwealth University, Biology Department, Box 842012, Richmond, VA 23284-2012, USA

Abstract. Src family protein tyrosine kinases (SrcPTK) play a central role in immunoglobulin E (IgE)-mediated activation of mast cells. Functional coupling of the high-affinity IgE receptor (FceRI) is initiated by the SrcPTK family member, Lyn, through an antigen aggregation-dependent transphosphorylation. Because Lyn is the ‘initiating’ kinase, an essential role in mast cell effector function was conferred. Recent studies challenge this view. Evidence demonstrating that Lyn kinase is dispensable for mast cell degranulation is now available. In contrast, another SrcPTK family member, Fyn, is required for degranulation and cytokine production. New studies, on mast cells expressing FceRIb ITAM mutants, show that the loss of Lyn interaction with FceRI has only a modest inhibitory effect on mast cell degranulation and an enhancing effect on lymphokine production, although many of the biochemical signals (including FceRI phosphorylation) were significantly impaired. In vivo studies on Lyn-null mice also demonstrated that this kinase is a negative regulator of IgE production and anaphylaxis, whereas Fyn kinase is required for anaphylaxis but not for IgE production. Collectively, these studies argue that sustained Lyn kinase activity negatively regulates mast cell responses. This suggests the possible existence of Lyn polymorphisms that may contribute in allergic disease. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 39–53

Recognition of an allergen by cell surface-bound IgE antibodies induces aggregation of FceRI on the surface of a mast cell or basophil, which elicits the release of preformed and de novo synthesized mediators of allergy and inflammation. The engagement of FceRI on the extracellular aspect of the cell membrane is interpreted intracellularly through multiple biochemical events that must occur before the mast cell can fully respond. Phosphorylation of proteins at tyrosine residues is 1

This paper was presented at the symposium by Juan Rivera to whom correspondence should be addressed. 39

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the first and key modification that governs the process of mast cell activation (Benhamou et al 1990). The Src protein tyrosine kinase (PTK) Lyn was demonstrated to associate with FceRI and cause its phosphorylation (Eiseman & Bolen 1992) (Pribluda et al 1994). The FceRI on mast cells and basophils is a tetrameric receptor with an IgE-binding a chain, a signal amplifying b chain, and two signal transducing disulfide-linked g-chains. Signalling competence is imparted by the presence of an immunoreceptor tyrosine-based activation motif (ITAM), on the b and g subunits, which contains tyrosine residues that can be modified by phosphorylation. Once phosphorylated by Lyn, the ITAMs become docking sites for other signalling proteins that amplify and sustain the biochemical signals that drive the cells response (reviewed in Nadler et al 2000). Obviously, Lyn kinase occupies an important initiating step in FceRI functional coupling. Therefore, an essential role for Lyn kinase in the release of inflammatory mediators has been conferred and the view that it may serve as a reasonable therapeutic target in allergic disease has been justifiably proposed (Metzger 1999). Nonetheless, the studies of Nishizumi and colleagues (Nishizumi & Yamamoto 1997) cast doubt on the relative importance of Lyn kinase in mast cell degranulation and cytokine production. Mast cells derived from mice with a homozygousnull mutation of the Lyn gene were not impaired in degranulation nor in cytokine production. In contrast, the previous report of a defective passive cutaneous anaphylaxis response observed in another Lyn-/- mouse (Hibbs et al 1995), suggested the critical need for Lyn kinase in mast cell degranulation. Thus, these contradictory data needed to be reconciled. Over the last several years, we and others (Kawakami et al 2000) have investigated the role of Src PTKs in mast cell activation and responses. In the course of these studies we discovered that another Src PTK, Fyn, was activated upon FceRI engagement. Mice or mast cells deficient in this kinase showed an ablated anaphylaxis or degranulation response, respectively (Parravicini et al 2002). These findings have led to a new paradigm in the functional coupling of FceRI (Rivera 2002, Blank & Rivera 2004). However, the relative importance of Src PTKs Fyn and Lyn to the allergic response remained unclear. Herein, we summarize our efforts of the past two years to address this issue through use of animal models. Association of Lyn and Fyn with FceRI and relative roles in receptor phosphorylation Considerable information has accumulated on the interaction of Lyn kinase with FceRI. This interaction is known to occur with the FceRIb subunit and is mediated, at least in part, through the unique domain of Lyn (Kihara & Siraganian 1994, Vonakis et al 1997), although upon FceRI stimulation further recruitment of Lyn is observed (Yamashita et al 1994) that is likely mediated through its SH2 domain

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and requires the phosphorylated canonical tyrosines of the FceRIb (Kihara & Siraganian 1994). FceRI-associated Lyn kinase appears to be fully active since its specific activity does not increase upon receptor stimulation (Pribluda et al 1994, Yamashita et al 1994). Unlike Lyn, Fyn activation requires FceRI stimulation ( Parravicini et al 2002) with an increase of its total specific activity by approximately threefold (Odom et al 2004). Preliminary experiments in which the tyrosine residues in the ITAM of the FceRIb were mutated, showed that the normal activation of Fyn requires an intact FceRIb ITAM (Y. Furumoto and J. Rivera, unpublished observation). Coimmunoprecipitation experiments demonstrated the specific association of Fyn with FceRI (Parravicini et al 2002). However, to date we have no convincing evidence of a direct interaction of Fyn with FceRIb or g. Nonetheless, Fyn has the potential to associate with antigen receptors since studies in T cells showed it associates with the T cell receptor z chain (a homologue of the FcRg ) through SH2 domain interactions (van’t Hof & Resh 1999). While we previously reported that FceRI phosphorylation was ablated by Lyndeficiency (Kovarova et al 2001), more sensitive methods of detection have allowed detection of phosphorylation at a level that is approximately 5–15% of wild-type cells. This minimal level of phosphorylation is maintained if both Lyn and Fyn are absent (G. Gomez, S. Odom, J. Rivera, unpublished observation). Consistent with this finding is the observation that mutation of the canonical tyrosines of the FceRIb ITAM, which led to a loss of Lyn, did not cause a complete loss of g chain phosphorylation (Furumoto et al 2004). Interestingly, while Lyn association was undetectable, only a modest loss of degranulation was observed. Amplification of signalling by Fyn and Lyn The events following Lyn-dependent phosphorylation of FceRI are well characterized and reviewed in detail (Kinet 1999). Briefly, Lyn is required for full activation of Syk kinase because phosphorylation of FceRI creates the phospho-ITAMs required for Syk interaction and activation. Syk phosphorylates multiple proteins but one key molecule that is a target of Syk activity is the linker for activation of T cells (LAT), which scaffolds a macromolecular signalling complex that includes proteins like PLCg, SLP-76 and Vav1 (Zhang et al 1998, Saitoh et al 2000, Saitoh et al 2003). These proteins play a key role in the regulation of calcium responses upon FceRI stimulation. In contrast, Fyn does not appear to play a major role in the regulation of intracellular calcium since Fyn-/- mast cells have normal LAT phosphorylation and apparently intact calcium responses (Parravicini et al 2002). Instead, the activity of phosphatidylinositol-3-kinase (PI3K) is impaired and both PDK1 activity and Akt phosphorylation are defective (Parravicini et al 2002). It is also important to note, possibly because of the role of PDK1 in activation of PKC,

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that PKC activity is reduced in these cells (Le Good et al 1998). This may account, in part, for the defective degranulation phenotype of Fyn-deficient mast cells as PKC activation is required for this event (reviewed in Rivera & Beaven 1997). The cause for the defect in PI3K activity is not completely clear but it is likely to be mediated through Fyn-dependent phosphorylation of Gab2, an adapter molecule that regulates PI3K activity in mast cells (Gu et al 2001). Importantly, it appears that the Lyn/LAT and Fyn/Gab2 pathways function in parallel but are complementary. In the absence of Lyn, Fyn is functional and PI3K activity (as measured by Akt phosphorylation) appears intact (Parravicini et al 2002). Thus, these cells can degranulate. Nonetheless, both the intracellular calcium chelator, BAPTA-AM, or the PI3K inhibitor, LY294002, effectively abrogate degranulation and/or cytokine responses of Lyn- or Fyn-deficient mast cells demonstrating the presence of residual calcium mobilization or PI3K activity, respectively, in these cells. Thus, both pathways seem necessary for normal mast cell activation (Parravicini et al 2002). Positive and negative regulation of mast cell responses by Lyn and Fyn kinases Evidence that Lyn dissociates from FceRI after receptor stimulation, and that signalling-competent receptors are devoid of Lyn and instead complex with Syk, has indirectly suggested that Lyn may play a negative regulatory role in mast cell responses (Ortega et al 1999) (Lara et al 2001). As mentioned above, Lyn-deficient mast cells degranulate. Thus, a reasonable hypothesis is that Lyn kinase is not the driving force for mast cell degranulation and may function to impair mast cell responses. A recent series of observations using Lyn-deficient mice and mast cells demonstrated this to be the case and defined a mechanism by which Lyn kinase controls mast cell degranulation (Odom et al 2004). The findings point to the loss of negative regulatory control of Fyn kinase activity in Lyn-deficient mast cells as key for the hyperdegranulation observed in these cells. Fyn is hyperactivated because Lyn is required for the phosphorylation of an adapter protein Csk-binding protein (Cbp), which functions to target the negative regulatory kinase, C-terminal Src kinase (Csk), whose function is to down-regulate Src PTK activity (Okada et al 1991); reviewed in (Lindquist et al 2003). The consequence is a selective and constitutive elevation of Fyn kinase activity in the absence of Lyn (Odom et al 2004). This enhanced Fyn kinase activity is contributory to the hyper-degranulation phenotype of Lyn-deficient mice and mast cells since genetic deletion of Fyn or of both Lyn and Fyn (Lyn/Fyn-/- double deficient) caused a 70–90% inhibition of mast cell degranulation. Moreover, unlike Lyn-deficient mice, Lyn/Fyn double-deficient mice were resistant to a systemic anaphylactic challenge (Odom et al 2004). The negative and positive roles for Lyn and Fyn, respectively, can also be demonstrated at the level of gene expression. Previous studies showed that Lyn-deficient


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mast cells have an intact cytokine secretory response (Nishizumi & Yamamoto 1997, Kawakami et al 2000). Our recent unpublished work also demonstrates that the absence of Lyn, in mast cells, causes increased expression of cytokine and chemokine mRNA (G. Gomez, C. Gonzalez-Espinosa and J. Rivera, unpublished results). Moreover, mutation of the FceRIb ITAM, which leads to the loss of receptor-associated Lyn and to decreased SHIP-1 phosphorylation, also resulted in increased cytokine production and secretion (Furumoto et al 2004). In contrast, the loss of Fyn caused a defect in the accumulation of interleukin (IL)6 and tumour necrosis factor (TNF)a mRNA (among others) that is mirrored by decreased secretion of these cytokines. While many transcription factors contribute to the induction of cytokine genes, we found that Fyn-deficiency caused a marked reduction in nuclear NF-kB activity whereas preliminary experiments showed that Lyn-deficient mast cells are less impaired than Fyn-deficient mast cells in nuclear NF-kB activity (G. Gomez, C. Gonzalez-Espinosa and J. Rivera, unpublished results). Moreover, the expression of the FceRIb ITAM-null mutant receptor led to a dramatically enhanced nuclear NF-kB activity that mirrored the increased cytokine production (Furumoto et al 2004). While it is clear that NF-kB alone does not account for the robust induction of cytokines in Lyn-deficient or FceRIb ITAM-null mast cells, the findings reflect the dichotomy of Lyn and Fyn influence at the transcriptional level. Thus, the view of negative and positive regulatory roles for Lyn and Fyn in mast cells, respectively, is upheld in the de novo induction of gene expression. The role of Lyn and Fyn in an allergic model The in vivo consequence of Lyn- or Fyn-deficiency in the passive systemic anaphylaxis model of allergy was explored. Lyn-deficient mice sensitized with dinitrophenyl (DNP)-specific IgE were subsequently challenged intravenously with DNP-human serum albumin (the latter was used as a carrier). When compared to wild type mice, Lyn-deficient mice, of 4 –5 weeks of age, showed increased responses as determined by measurement of circulating histamine (Odom et al 2004). In contrast, Fyn-deficient mice were resistant to anaphylaxis. On average the levels of circulating histamine were less than 20% of those seen in wild-type mice. This in vivo challenge of mast cells strongly supports the negative or positive role for Lyn and Fyn, respectively, in mast cell degranulation. As mentioned above, Hibbs and colleagues found Lyn-deficient mice to be resistant to a passive cutaneous anaphylactic challenge (Hibbs et al 1995). While this model measures the response of localized skin mast cells, one might expect similar results in both models. This led us to investigate the anaphylactic response of these mice at various ages, since this was one variable between the studies. Interestingly, as age increased so did resistance to anaphylaxis (Odom et al 2004). Because increased B cell sensitivity to IL4-mediated immunoglobulin class switching had been described, we

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tested the IgE levels of young (8 weeks). Normal levels of IgE were observed in young mice whereas older mice showed increased (up to 20-fold greater) levels of circulating IgE. Mast cells from the peritoneum of these mice showed increased FceRI expression and almost complete saturation of receptors with IgE (Odom et al 2004). The availability or full occupancy of receptors with IgE is the likely explanation for the apparent hyper- and hypo-anaphylactic response of young and old Lyn-deficient mice, respectively. As mice age, passive in vivo sensitization of mast cells with a DNP-specific IgE is not possible and thus older mice do not respond to the DNP challenge. The increase in circulating IgE coupled with the hyperresponsive nature of mast cells in Lyn-deficient mice led us to explore the in vivo consequences of this phenotype. We found that unchallenged Lyn-/- mice had at least a twofold higher level of circulating histamine than wild-type mice (Odom et al 2004). In addition, the numbers of mast cells in the skin and peritoneum were increased by at least twofold and these mast cells expressed higher amounts of FceRI. A peritoneal eosinophilia was also found consistent with the presence of activated mast cells in this compartment. In contrast, Fyn-/- mice showed normal mast cell numbers in the peritoneum, no eosinophilia and, unless challenged, circulating histamine levels were identical to unchallenged wild-type mice. Moreover, Fyn-/- mice had normal serum IgE levels regardless of age. Thus, the findings argue for a link between the increase in circulating IgE levels, hyperresponsive mast cells, and the allergic-like phenotype in Lyn-/- mice. Importantly, mast cells from these mice showed increased FceRI expression, a feature demonstrated to increase the sensitivity of a mast cell to allergen challenge (Yamaguchi et al 1997, 1999). The previous in vitro observation that very high levels of monomeric IgE can induce mast cell degranulation through aggregation of FceRI may be recapitulated in the Lyn-/- mice (Kitaura et al 2003). SHIP-1-/- mice also show a hyperresponsive mast cell phenotype and high susceptibility to monomeric IgE-dependent degranulation (Huber et al 1998). Therefore, the phenotype of high circulating IgE levels combined with the hyperresponsive mast cell phenotype may be key in the allergic-like phenotype of Lyn-/- mice. This possibility must be formally tested but is a plausible explanation for the high (all mice) occurrence of the allergic-like phenotype in these mice. Closing remarks In summary, we provide evidence for the functional coupling of two Src PTKs (Lyn and Fyn) in response to FceRI stimulation. Our findings suggest that, beyond its initial role in FceRI phosphorylation, Lyn kinase primarily serves as a negative regulator of both mast cell degranulation and cytokine production. Importantly, our findings also provide preliminary evidence for FceRI phosphorylation in the


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absence of both Lyn and Fyn. This implies molecular redundancy in this initial step provided by another kinase(s) and suggests the possibility that FceRI may be a target of multiple kinases, possibly even in the presence of Lyn kinase. Interestingly, our studies show that Lyn functions in a receptor-dependent and -independent manner to regulate mast cell degranulation. Thus, mutation of the FceRIb ITAM, and loss of Lyn association, had a modest inhibitory effect on degranulation. In contrast, the absence of Lyn from mast cells enhanced degranulation. Thus, the negative regulation of mast cell degranulation by Lyn is independent of its association with FceRI. However, this dichotomy does not extend to cytokine production since both Lyn-deficient mast cells and FceRIb ITAM-null mutant mast cells showed increased cytokine production, arguing for dependence on FceRIb-associated Lyn. Our findings also demonstrate that Fyn kinase is a positive modulator of mast cell degranulation and cytokine production. Fyn drives the hyperdegranulation phenotype of Lyn-deficient mast cells but is not responsible for the increased IgE production of Lyn-deficient B cells. Thus, how Lyn functions to regulate IgE production is of considerable interest. Our studies to date were conducted with murine models. We do not know if Fyn and Lyn kinases, which are expressed in human mast cells (Blank & Rivera 2004), have the same functional role as observed in mice. Nonetheless, based on correlative studies, decreased expression of Lyn kinase in B cells has been associated with systemic lupus erythematosus (Liossis et al 2001) suggesting an in vivo role in tolerance. The molecular consequence in the loss of normal levels of Lyn expression in these patients is unclear. However, the apparent negative regulatory control of Lyn on immunoglobulin production and class switching make the issue of autoantibody production of particular interest. It also brings to bear the possibility that yet to be identified Lyn polymorphisms might be associated with atopy and allergy. Acknowledgements The Molecular Inflammation Section receives support from the Department of Health and Human Services, National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases.

References Benhamou M, Gutkind JS, Robbins KC, Siraganian RP 1990 Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release. Proc Natl Acad Sci USA 87:5327–5330 Blank U, Rivera J 2004 The ins and outs of IgE-dependent mast cell exocytosis. Trends Immunol 25:266–273 Eiseman E, Bolen JB 1992 Engagement of the high-affinity IgE receptor activates src proteinrelated tyrosine kinases. Nature 355:78–80

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Furumoto Y, Nunomura S, Terada T, Rivera J, Ra C 2004 The Fcepsilon RIbeta exerts inhibitory control on MAP kinase and Ikappa B kinase phosphorylation and mast cell cytokine production. J Biol Chem 279:49177–49187 Gu H, Saito K, Klaman LD et al 2001 Essential role for Gab2 in the allergic response. Nature 412:186–190 Hibbs ML, Tarlinton DM, Armes J et al 1995 Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 83:301–311 Huber M, Helgason CD, Damen JE et al 1998 The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation. Proc Natl Acad Sci USA 95:11330–11335 Kawakami Y, Kitaura J, Satterthwaite AB et al 2000 Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation. J Immunol 165:1210–1219 Kihara H, Siraganian RP 1994 Src homology 2 domains of Syk and Lyn bind to tyrosinephosphorylated subunits of the high affinity IgE receptor. J Biol Chem 269:22427–22432 Kinet JP 1999 The high-affinity IgE receptor (FceRI): From physiology to pathology. Annu Rev Immunol 17:931–972 Kitaura J, Song J, Tsai M et al 2003 Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the Fc(epsilon)RI. Proc Natl Acad Sci USA 100:12911–12916 Kovarova M, Tolar P, Arudchandran R et al 2001 Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fc epsilon receptor I aggregation. Mol Cell Biol 21:8318–8328 Lara M, Ortega E, Pecht I et al 2001 Overcoming the signaling defect of Lyn-sequestering, signal-curtailing FceRI dimers: aggregated dimers can dissociate from Lyn and form signaling complexes with Syk. J Immunol 167:4329–4337 Le Good JA, Ziegler WH, Parekh DB et al 1998 Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281:2042–2045 Lindquist JA, Simeoni L, Schraven B 2003 Transmembrane adapters: attractants for cytoplasmic effectors. Immunol Rev 191:165–182 Liossis SN, Solomou EE, Dimopoulos MA et al 2001 B-cell kinase lyn deficiency in patients with systemic lupus erythematosus. J Investig Med 49:157–165 Metzger H 1999 It’s spring, and thoughts turn to. . .allergies. Cell 97:287–290 Nadler MJ, Matthews SA, Turner H, Kinet J-P 2000 Signal transduction by the high-affinity immunoglobulin E receptor Fc epsilon RI: coupling form to function. Adv Immunol 76:325–355 Nishizumi H, Yamamoto T 1997 Impaired tyrosine phosphorylation and Ca2+ mobilization, but not degranulation, in Lyn-deficient bone marrow-derived mast cells. J Immunol 158:2350–2355 Odom S, Gomez G, Kovarova M et al 2004 Negative regulation of IgE-dependent allergic responses by Lyn kinase. J Exp Med 199:1491–1502 Okada M, Nada S, Yamanashi Y, Yamamoto H, Nakagawa H 1991 CSK: a protein-tyrosine kinase involved in regulation of src family kinases. J Biol Chem 266:24249–24252 Ortega E, Lara A, Lee I et al 1999 Lyn dissociation from phosphorylated FceRI subunits: A new regulatory step in the FceRI signaling cascade revealed by studies of FceRI dimer signaling activity. J Immunol 162:176–185 Parravicini V, Gadina M, Kovarova M et al 2002 Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol 3:741–748 Pribluda VS, Pribluda C, Metzger H 1994 Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation. Proc Natl Acad Sci USA 91:11246–11250 Rivera J 2002 Molecular adapters in FceRI signaling and the allergic response. Curr Opin Immunol 14:688–693


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Saitoh S, Arudchandran R, Manetz TS et al 2000 LAT is essential for Fc(epsilon)RI-mediated mast cell activation. Immunity 12:525–535 Saitoh S, Odom S, Gomez G et al 2003 The four distal tyrosines are required for LATdependent signaling in FceRI-mediated mast cell activation. J Exp Med 198:831–843 van’t Hof W, Resh MD 1999 Dual fatty acylation of p59(Fyn) is required for association with the T cell receptor zeta chain through phosphotyrosine-Src homology domain-2 interactions. J Cell Biol 145:377–389 Vonakis BM, Chen HX, Haleem-Smith H, Metzger H 1997 The unique domain as the site on Lyn kinase for its constitutive association with the high affinity receptor for IgE. J Biol Chem 272:24072–24080 Yamaguchi M, Lantz CS, Oettgen HC et al 1997 IgE enhances mouse mast cell FceRI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions. J Exp Med 185:663–672 Yamaguchi M, Sayama K, Yano K et al 1999 IgE enhances Fce receptor I expression and IgEdependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fce receptor I expression and mediator release. J Immunol 162:5455–5465 Yamashita T, Mao S-Y, Metzger H 1994 Aggregation of the high-affinity IgE receptor and enhanced activity of p53/56lyn protein-tyrosine kinase. Proc Natl Acad Sci USA 91:11251–11255 Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP, Samelson LE 1998 LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83–92

DISCUSSION Koffer: What happens to the mast cells in Fyn-deficient mice? Rivera: There is no defect in numbers of peritoneal mast cells, but we haven’t analysed skin mast cells. IgE levels are normal. Stevens: Have single nucleotide polymorphism (SNP) analyses been carried out on the human FYN and LYN genes? If so, has a SNP been identified in either gene that results in the expression of a defective protein? The FYN and LYN genes reside on human chromosomes 6q21 and 8q13, respectively. Thus, in a related question, has either site been linked with airway hyper-responsiveness or with any mast cell-dependent disorder in humans or mice? Rivera: To the best of my knowledge, no SNP analysis has been done, although this is underway. There is only one paper that is suggestive of a link between LYN expression and disease: this is from a group in Greece who demonstrated that reduced levels of LYN expression in B cells seemed to be associated with systemic lupus erythematosus (Liossis et al 2001). But they did not analyse this from the perspective of allergic individuals. Razin: You have shown that by sRNAi you can inhibit 80–90% of the expression of Lyn. However, you show that there is no correlation between phosphorylation and degranulation. Is 10–20% of the normal level of Lyn enough to be associated with the receptor and do the job? Rivera: The phenotype we see is a hyperdegranulation phenotype. Lowering of Lyn levels seems to cause higher degranulation. We haven’t looked at whether recep-

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tors in the human cells, where Lyn is knocked down, are being phosphorylated but this is something on the agenda. Our data in human and mice suggest that there is still some phosphorylation of the receptor taking place, even in the total absence of Lyn. This doesn’t seem to be mediated by Fyn, because when we knock out both kinases we still get receptor phosphorylation. We don’t know what kinase is responsible for this activity. Razin: Regarding the IgE, did you check whether Lyn is involved in IL4 production? Rivera: In the mast cell it is clear that Lyn has a regulatory control over IL4. Without Lyn we see higher levels of mast cell-derived IL4. It could be that the Lyndeficient mast cells are contributing to the sustained activation of B cells and thus hyper IgE production. If we isolate the populations of B cells and mast cells, the B cells are hyper-responsive to IL4 in the absence of Lyn. In vivo, it remains to be determined where the IL4 is coming from, but clearly there is a defect in the B cells’ response to IL4. MacGlashan: I don’t like to look at the response of these cells by looking at an antigen concentration dependence-type curve, because the dynamics at different antigen concentrations lead to some interesting results. Rather, given that we are looking at a balance of positive and negative influences mediated by Lyn and Fyn, I wonder whether you have done an experiment where you titrate in a certain amount of IgE and measure a response. This might give sensitivity shifts that are dramatic. You are challenging with the same antigen so it fixes the response. Rivera: Those experiments have been done. We see the same phenotype. Even at low levels of receptor occupancy with IgE you get a shift of the response of Lyndeficient cells to the left, as compared with the wild-type cells. If we stimulate the cells with pharmacological agents, it is interesting to note that the Lyn-deficient cells showed a much higher response under those conditions than wild-type cells. It does suggest a dysregulation of the pathways leading to degranulation. MacGlashan: Are there strain differences in how this whole balance works? Rivera: That’s a good question, which we got from a number of reviewers! We are in the process of backcrossing these mice into the Balb/c background; we have so far been looking at mice in the C57/BL6 background. The initial studies were done with mice backcrossed four times. We now have mice that have been backcrossed six times to C57/BL6. They are showing the same phenotype. Koyasu: From your Fyn knockout phenotype it seems that the contribution of Fyn is substantial. When you see the LAT-deficient mast cells, the contribution also seems to be very high. In the absence of LAT, is the inhibitory effect of Lyn enhanced and therefore do you see lower activation of the Fyn pathway? You showed that the NF-kB pathway is mostly mediated by the Fyn pathway. Could NF-kB be involved in the LAT pathway?


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Rivera: I can only partially answer these questions based on the limited experiments we have done to date. With regards to the relative contribution of Fyn and LAT to degranulation, our recent findings looking at Fyn/LAT double-deficient mice suggest that there is a contributory role mediated by both since mast cell responses are further reduced. In terms of NF-kB, the only thing I can say is that at least in the LAT-deficient mast cells, NF-kB can still be activated, although at least in preliminary experiments this looks partially defective. On the other hand, in Lyn-deficient mast cells this looks pretty normal. I am not quite sure where the connection to NF-kB is actually occurring, although Fyn seems to contribute to NF-kB activation. This is an important question because we also don’t know whether Fyn and LAT communicate with each other. Marshall: What do you think is the basis for the eosinophilia you see? Is there a peripheral blood eosinophilia? Do you see increased neutrophils in the peritoneum? Rivera: We have looked in the bone marrow and we see normal numbers of eosinophils. It is a bit difficult for us at this stage to provide a good answer. We are looking to see whether this is mast cell driven as a consequence of mast cell production of eotaxin. It could also be Lyn driven. Eosinophils express Lyn, which might have a negative regulatory restraint on eosinophil proliferation. We are trying to address these possibilities at the moment. Marshall: You expressed it as a percentage of peritoneal cells. In terms of total numbers is the increase similar? Rivera: Yes. Ono: You said in SHIP-deficient mice, NF-kB is elevated. What do you mean by this? Rivera: This is not our work; it is from Gerry Krystal’s lab (Kalesnikoff et al 2002). They see activation of IKKb in SHIP-deficient mast cells. They can detect p65Rel in the nucleus. We also detect p65Rel but were unable to detect p50. I think this may be an issue of whether this subunit is actually utilized in the mast cell system. Ono: What effect does cytokine secretion have on mast cell survival? Rivera: We don’t know. Kawakami: The increased Fyn activity in Lyn knockout mast cells is very interesting. What do you think about the mechanism underlying this phenomenon? Rivera: We have been able to demonstrate that there is a dysregulation of the basic mechanism involved in regulating Src family kinases. The negative regulatory tyrosine at position 527 is not being phosphorylated due to the loss of Csk (Cterminal Srk kinase) targeting to the membrane. Essentially, Csk is targeted to the membrane through an interaction with an adaptor protein called Csk binding protein (Cbp). This needs to be tyrosine phosphorylated in order for Csk to target to the membrane and act on Src kinases. In the absence of Lyn you lose that tyrosine phosphorylation of the Csk binding protein. You don’t target Csk to the membrane thus failing to down-regulate Fyn activity.

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Kawakami: So is Fyn the kinase that phosphorylates Cbp/PAG? Rivera: No, Lyn is the kinase that phosphorylates Cbp/PAG and then Csk is brought to the membrane and this phosphorylates Fyn at the 527 position. We can demonstrate this by reconstitution experiments. If we put Lyn back into the cells we can demonstrate that we can now target Csk to the membrane, and can now phosphorylate Fyn and down-regulate its activity. The hyper responsive degranulation phenotype that one sees in the Lyn-deficient cell is returned to normal levels. Kawakami: There is one paper published a few years back (Ohtake et al 2002) on the kinetics of Cbp/PAG phosphorylation after FceRI cross-linking. In this paper they showed increased tyrosine phosphorylation in Cbp after the cross-linking. Have you studied this? Rivera: We have looked at Lyn-deficient cells, reconstituted them, and compared these to wild-type and Fyn-deficient cells. Fyn-deficient and wild-type cells have a normal mechanism of Csk targeting to the membrane via Cbp. It gets phosphorylated normally and is targeted to the membrane. We have to realize that Cbp is phosphorylated by Lyn under basal conditions in resting cells. Thus, there is always some Csk that is targeted to the membrane in resting cells. Then, as described by Yamashita and colleagues (Ohtake et al 2002), its membrane targeting is further enhanced by the increased phosphorylation of Cbp. We think that loss of that basal mechanism in the absence of Lyn leads to the enhanced constitutive Fyn activity we see in the cells. Presumably, what happens in wild-type cells is that as more Csk is recruited, Fyn activity is down-regulated as a mechanism to control the amount of Fyn that is activated and to regulate the degranulation response. In the absence of Lyn this mechanism simply isn’t present and there is a hyperdegranulation response. I think this is why reconstitution experiments are key. We can show that this mechanism can be entirely reconstituted by putting wild-type Lyn back into Lyn-deficient cells. Ono: Have you looked at the effects on receptor cross-talk in the bITAM mutants? For example, what effect is there on the cross-talk between IgE receptor and chemokine receptors, and is there a proportional decrease? I ask this because of the considerable evidence now that G protein-coupled receptors are likely to be critical for mast cell degranulation in vivo? Rivera: We haven’t, but it’s an interesting idea. There is preliminary circumstantial evidence suggesting that in the absence of Lyn, receptor cross-talk may be a possibility. MacDonald: Would you predict that you would see the same thing if you did this in an inducible knockout rather than a constitutive one? Rivera: The first step would be to create a mast cell Cre mouse, which a number of groups are working on. This remains to be seen, but it is likely that this would


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be the case. This is suggested by the shRNA approach we have used in the human system, which seemingly recapitulates the murine data. Stevens: In collaboration with Dr Adachi (MD Anderson Cancer Center, Houston, TX), we recently created the ‘mMCP-5-promoter Cre’ mouse that should enable one to evaluate the mast cell contribution of an ubiquitously expressed gene (e.g. LYN or SYN) in vivo. The mMcp5 gene is selectively expressed in mast cells (McNeil et al 1991), and we discovered that the most critical cis-acting elements that regulate its transcription reside in its 5¢ flanking region rather than its introns, exons, or 3¢ flanking region. Using a homologous recombination approach, we placed the Cre recombinase gene immediately downstream of the translation-initiation site of the mMcp5 gene. The mMCP-5-promoter-Cre mouse should be invaluable because it can be mated with a transgenic mouse strain that has a floxed gene that encodes a protein that normally is ubiquitously expressed in the body to selectively diminish its levels in mast cells. One can even use the mouse strain to selectively express a foreign gene in mast cells. These possibilities are presently being evaluated by mating our mMCP5-promoter-Cre animals with the B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J mouse strain (Jackson Labs) that constitutively express lacZ under the control of the CMV enhancer/chicken actin promoter. When crossed with a Cre-expressing strain such as the one we created, lacZ expression is replaced with enhanced green fluorescent protein (GFP) in those cells that are expressing Cre. This double reporter system makes it possible to distinguish a lack of reporter expression from a lack of Cre recombinase expression while providing a means to assess Cre excision activity in living animals and their cells. MacGlashan: I would like to hear Henry Metzger and Juan Rivera talk about titrations with different levels of Lyn. How does this fit in with the fact that Lyn looks like an inhibitory signal? Metzger: I don’t have an easy answer for that. I didn’t ask Juan Rivera this question because I have asked him about it several times before. My impression is that he still isn’t clear as to what the molecular mechanism is for how aggregation is activating Fyn. Rivera: That’s correct. We don’t have a clear understanding of how the receptor and Fyn communicate to each other. At least at this stage, our findings are consistent with regards to the initial event of receptor phosphorylation. It is clear in our data as well as Henry’s data that Lyn is important for the phosphorylation of the receptor. A second consideration that needs to be put into place here is that, without question, there are compensatory mechanisms in Lyn deficiency. Whether or not these compensatory mechanisms are overcoming a threshold limit for degranulation is a possibility that may be reflected by hyperactivation of Fyn kinase. Our data on the FceRIb ITAM mutants are also suggestive of compensation in the total absence of Lyn in that they demonstrate only a partial defect on degranulation when

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Lyn is lost from the receptor. Yet, on the other hand, cytokine production is dramatically enhanced under these conditions. The question becomes an issue of the therapeutic value in targeting Lyn or disrupting Lyn’s interaction with receptor. We don’t think it would be a good therapeutic target. Pecht: Could you elaborate on how the SH2 domain containing inositol 5¢ phosphatase (SHIP) gets involved in this? Rivera: All I can say is that in the absence of Lyn or in the absence of Lynmediated phosphorylation of receptor we see several things. The in vitro experiments show a loss of SHIP interaction with the receptor b chain itself. In the absence of Lyn or when the b chain is mutated, SHIP phosphorylation is reduced. We know that phosphorylation of SHIP is not required for its activity, but it is required for its association with Lyn kinase, through Lyn kinases SH2 domain. It could well be that we are seeing is a double whammy here: we are losing some or all of Lyn associated with receptor and the association of SHIP1 with Lyn. In this context we also see an increase of PIP3 in the cells expressing mutant receptors. This would suggest that the hyper-responsive cytokine activity may be coming from overproduction of PIP3. The increase in PIP3 is the phenotype of the Lyndeficient mast cells as well. Razin: I missed something regarding Fyn. In the Lyn knockout mice did you check whether there is acceleration of expression of Fyn, or whether there is enhancement in the activity of Fyn? Rivera: There is an enhancement of the activity of Fyn kinase in those cells. Interestingly there is also a slight increase in the amount of Fyn. We see about a 30% increase in protein. Razin: In your sRNAi experiments is there any enhancement of Fyn activity or expression? Rivera: We have gone through various Src family kinases in those experiments. Everything reflects the activity of wild-type cells except for that Src kinase which were are targeting. Razin: If you interfere with the balance of Fyn and Lyn, there is the essential role played by Lyn described by Henry Metzger. The problem is that when you reduce the amount of Lyn you increase the expression and activity of Fyn artificially. Rivera: Your point is well taken for the murine system; this is what we see in mice. But the point that Fyn is still the one that drives the degranulation response is answered by the double knockout. There we are actually generating a Lyn/Fyn double knockout and they become non-responsive. In the human system we have so far found that there doesn’t seem to be a compensatory mechanism with regards to Src family kinases. In preliminary experiments looking at signalling, there doesn’t seem to be a dysregulation of signalling pathways other than those that are targeted by the shRNA itself. If we look at Akt phosphorylation in Fyn knockdowns, we


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see a defect in Akt, which is what we see in the murine Fyn-deficient cells. This seems to be holding true, but we don’t see any compensatory mechanisms. References Kalesnikoff J, Baur N, Leitges M et al 2002 SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-kB activity. J Immunol 168:4737–4746 Liossis SN, Solomou EE, Dimopoulos MA, Panayiotidis P, Mavrikakis MM, Sfikakis PP 2001 B-cell kinase lyn deficiency in patients with systemic lupus erythematosus. J Investig Med 49:157–165 McNeil HP, Austen KF, Somerville LL, Gurish MF, Stevens RL 1991 Molecular cloning of the mouse mast cell protease-5 gene. A novel secretory granule protease expressed early in the differentiation of serosal mast cells. J Biol Chem 266:20316–20322 Ohtake H, Ichikawa N, Okada M, Yamashita T 2002 Transmembrane phosphoprotein Cskbinding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains as a negative feedback regulator of mast cell signaling through the FceRI. J Immunol 168:2087–2090

RasGRP4 in mast cell signalling and disease susceptibility Richard L. Stevens, Nasa Morokawa, Jing Wang* and Steven A. Krilis* Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA, and * Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital and Department of Medicine, University of New South Wales, Kogarah NSW 2217, Sydney, Australia

Abstract. The nucleotide sequences of the mouse, rat and human cDNAs and genes that encode the fourth member of the Ras guanine nucleotide releasing protein (RasGRP) family of signalling proteins have been deduced. RasGRP4 is a mast cell-restricted, cationdependent, guanine nucleotide exchange factor (GEF). It is also a diacylglycerol (DAG)/phorbol ester receptor that plays a prominent role in dictating which protease and eicosanoid mediators are expressed in rodent and human mast cell lines. RasGRP4 appears to act downstream of the tyrosine kinase receptor c-Kit/CD117 and upstream of the basichelix-loop-helix-leucine zipper transcription factor MITF. Allelic variants of RasGRP4 have been identified, as have functionally different isoforms that are the result of variable splicing of its gene. Earlier gene-linkage studies revealed a site on chromosome 7A3-B1 that controls intrinsic airway reactivity to methacholine in backcrossed C3H/HeJ and A/J mice. The 18-exon mouse RasGRP4 gene resides on chromosome 7A3-B1, and recent studies revealed that the mast cells developed from the hyporesponsive C3H/HeJ mouse strain preferentially produce a defective isoform of RasGRP4. These and other data suggest that RasGRP4 is of critical importance in mast cell development and that the expression of abnormal isoforms of the protein can lead to mast cell dysfunction. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 54–77

Identification of the genes and transcripts that encode Caenorhabditis elegans, mouse, rat and human RasGRPs Members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins (also known as GTPases) play pivotal roles in virtually all cells. Just as important are the guanine nucleotide exchange factors (GEFs) that activate each GTPase. For example, CDC25 is a Ras-specific GEF that is essential for the viability of Saccharomyces cerevisiae ( Jones et al 1991). The RasGRP2 gene (also known as CalDAGGEF1) was first identified in 1997 when Kedra and co-workers sequenced a cancer-susceptibility region on human chromosome 11q13 (Kedra et al 1997). The following year, cDNAs that encode RasGRP1, RasGRP2 and RasGRP3 were iso54

RasGRP4 REGULATES MAST CELL DEVELOPMENT AND FUNCTION

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lated from the brain when three groups independently attempted to identify signalling proteins that might be important in neuron development and/or function (Ebinu et al 1998, Kawasaki et al 1998, Nagase et al 1998). Although the amino acid sequences of RasGRP1, RasGRP2 and RasGRP3 are closely related, these proteins and their transcripts reside at different locations in the brain. Thus, the RasGRPs are functionally distinct. Subsequent transcript profiling studies (see UniGene Clusters Hs.189527, Hs.99491 and Hs.24024 at GenBank Locus ID sites 10125, 10235 and 25780) revealed the surprising finding that no RasGRP transcript is abundant in human brain (Table 1). Rather, RasGRP1 is preferentially expressed in the thymus; RasGRP2 in the peripheral blood; and RasGRP3 in the lymph node. It is now known that T cells express RasGRP1, megakaryocytes and platelets express RasGRP2, B cells express RasGRP3, and that the RasGRP family of signalling proteins is required for the development of a fully functional immune system. The GEFs that were identified in mast cells in the 1990s (e.g. Sos-1 [Turner et al 1995] [Table 1] and Vav1 [Song et al 1996, 1999]) are widely expressed. Thus, the GEFs that act downstream of the plasma membrane receptor c-Kit/CD117 and upstream of the intracellular transcription factor MITF to preferentially induce the development of mast cells from their multipotential progenitors remained to be identified (Fig. 1). Mouse bone marrow-derived mast cells (mBMMCs), developed with interleukin (IL)3 in the presence or absence of c-Kit ligand (KitL)/ stem cell factor, are immature mast cells that give rise to phenotypically different populations of mature mast cells when adoptively transferred into mast celldeficient WBB6F1-KitW/KitW-v (W/W v ) mice. Because these non-transformed mast cells must contain one or more GEFs that control the intermediate to final stages of mast cell development, thousands of clones were arbitrarily sequenced from a BALB/c mBMMC cDNA library in 1999. That transcript hunt resulted in the isolation of a novel cDNA that encoded the fourth member of the RasGRP family of GEFs (Yang et al 2002). No other RasGRP transcript was detected in mBMMCs. In addition, RasGRP4 was not detected at the mRNA or protein level in the mouse brain or in those cell types that express RasGRP1, RasGRP2 or RasGRP3. Thus, RasGRP4 is not coordinately expressed with any of its family members. The mouse RasGRP4 cDNA was used in a homology-based cloning approach to isolate its rat and human orthologues (Li et al 2002, 2003a, Yang et al 2002). Every rat, mouse and human mast cell analysed to date expresses RasGRP4. In contrast, the levels of the RasGRP4 transcript were below detection in all examined mouse myelomonocytic/macrophage, fibroblast, epithelial and T cell lines. Kinetic analysis of the RasGRP4 transcript levels in mBMMC cultures revealed that this signalling protein is expressed at the 2 week time point before most of the cells in the cultures become granulated (Li et al 2003a). At the protein level, the normal non-transformed mature mast cells that reside in the tongue, skin and peritoneal

TABLE 1

Expression of human GEFs

Tissue

Sos-1

RasGRP1

RasGRP2

RasGRP3

RasGRP4

bladder bone bone marrow brain cervix colon eye heart kidney larynx liver lung lymph node mammary gland muscle ovary pancreas periph. nerve placenta prostate skin small intest. soft tissue spleen stomach tongue testis thymus uterus vascular blood

0 53 54 67 169 70 43 107 74 41 30 31 163 140 73 10 12 119 64 77 66 0 153 180 9 291 15 0 46 115 66

0 0 0 13 0 0 74 0 22 0 0 0 187 0 18 31 49 0 17 15 0 0 0 60 9 0 30 444 17 0 0

0 0 164 54 0 11 0 0 0 0 7 3 62 0 18 0 12 0 17 31 6 0 0 0 0 0 38 0 11 38 237

0 17 109 37 0 5 12 0 52 41 7 28 280 8 45 0 0 0 30 0 18 0 0 0 19 72 15 0 23 77 39

0 0 136 2 0 0 0 0 37* 0 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 52

Noted above is the estimated number of each GEF transcript/million transcripts in various human tissues. The data were calculated based on the number of dbESTs in GenBank’s database as of October 2004. The obtained data are from UniGene Clusters Hs.326392, Hs.189527, Hs.99491, Hs.24024 and Hs.130434. For reference, the level of the glyceraldehyde-3-phosphate dehydrogenase transcript varies from 666–10 302 transcripts/million transcripts. The RasGRP4 gene and the gene that encodes the hypothetical protein LOC147965 are orientated back-to-back on chromosome 19q13.1. Because the two genes overlap by 28 nucleotides, some of the dbESTs in GenBank that previously were assigned to the RasGRP4 gene (e.g. accession numbers AA425337 and AA490327) actually originated from the adjacent LOC147965 gene. Moreover, some of the dbESTs in the database encode dysfunction isoforms of RasGRP4. The accumulated data suggest that the expression of functional RasGRP4 is even more restricted than that indicated in the above profiling study. * The kidney RasGRP4 dbESTs in the above profiling study were isolated from a clear cell tumour; there is no evidence that RasGRP4 is expressed in a normal kidney.


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KitL

c-kit /CD117

Mast Cell Progenitor

? Ras GDP

Gene Activation

Ras GTP

MITF Hint MITF

Granule Proteases PGDS

FIG. 1. Signal transduction in c-Kit-activated MCs and their progenitors. Circulating mast cellcommitted mononuclear progenitors encounter KitL and other differentiation factors when they enter tissues. Binding of KitL to its tyrosine kinase receptor c-Kit/CD117 activates a poorly defined signalling cascade (black box) which eventually causes activation of Ras and other GTPases. Activated GTPases induce the dissociation of MITF/HINT complexes in the cytosol. Liberated MITF translocates into the nucleus where this DNA-binding protein induces the transcription of the genes that encode PGD2 synthase and numerous mast cell-restricted granule proteases.

cavity of the BALB/c mouse contain high levels of RasGRP4 protein, as do the v-abl-expressing mast cells that develop in the spleen of the V3 mastocytosis mouse. Although RasGRP4 is expressed in mast cell-committed progenitors, this signalling protein continues to be expressed in mast cells as these immune cells complete their differentiation and granule maturation in tissues. As assessed by RNA blot, RT-PCR, immunohistochemistry and SDS-PAGE/ immunoblot analysis, RasGRP4 is a mast cell-restricted intracellular protein in humans. As in the mouse, RasGRP4 is also expressed in the circulating mononuclear progenitors that give rise to mature human mast cells (Table 1). The non-transformed human mast cells that are obtained by culturing cord blood progenitors in the presence of IL6, IL10 and KitL also express RasGRP4 (R.L. Stevens, J. Boyce, and S.A. Krilis, unpublished observation). As assessed immunohistochemically, no mature lymphocyte, platelet, macrophage, neutrophil, or eosinophil has been identified in a normal human or mouse tissue that contains appreciable amounts of RasGRP4 protein. Because mononuclear phagocytes and mast cells originate from a common CD34+ progenitor, the failure to detect immunoreactive RasGRP4 protein in tissue macrophages suggests that this intracellular signalling

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protein ceases to be expressed when circulating multipotential CD34+ progenitors are induced to become mature tissue macrophages. In support of the conclusion that the levels of RasGRP4 can be reversibly regulated in haematopoietic progenitors after they exit the bone marrow, mononuclear cells in human peripheral blood quickly cease expressing human RasGRP4 mRNA when they are exposed to lectins (Yang et al 2002). The human RasGRP4 gene is ~17 kb in size, consists of 18 exons, and resides on chromosome 19q13.1 at a site the Human Genome Consortium was having difficulty sequencing (Yang et al 2002). For some unknown technical reason, the Mouse Genome Consortium still has been unable to deduce the nucleotide sequence of the 5¢ flanking region and the 5¢ untranslated region of the mouse RasGRP4 gene. The RasGRP1, RasGRP2 and RasGRP3 genes reside on human chromosomes 15q15, 11q13 and 2p25.1, respectively. The corresponding mouse genes reside on chromosomes 2, 19 and 17; the mouse RasGRP4 gene resides on chromosome 7. Thus, no two RasGRP genes are physically linked in the genome. The nematode C. elegans lacks T cells, B cells, platelets and mast cells. Nevertheless, a BLASTp search of the WormBase database (http://www.wormbase.org) revealed that the C. elegans gene F25B3.3 on chromosome V encodes the putative 654-residue protein WP:CE34003 that is ~40% identical to RasGRP1, RasGRP2, RasGRP3 and RasGRP4 (Fig. 2). The F25B3.3 gene has not been described in a publication. However, the accumulated data suggest that the F25B3.3 gene duplicated multiple times before the evolution of mammals. The resulting four genes translocated to distinct chromosomes and then underwent substantial nucleotide and amino acid divergence to create the final RasGRP1, RasGRP2, RasGRP3 and RasGRP4 genes in mice and humans which were then used to control the development and function of our immune cells. All four RasGRP genes were then maintained throughout the last 100 million years of evolution. The fact that the four RasGRP signalling proteins appeared quite early in evolution was the first indication of their importance. Analysis of the varied domains in RasGRP4 Each RasGRP is a modular protein that contains a Ras exchanger motif, a CDC25like catalytic GEF domain, a DAG/phorbol ester-binding domain, and at least one EF-hand motif that the protein uses to bind Ca2+ ions. The major RasGRP4 isoform expressed in normal human and mouse MCs is ~75 kDa and consists of 673 amino acids. The predicted 3D structures of residues 34 –445 and 541–597 in mouse RasGRP4 are shown in Fig. 3. RasGRP4 is 36–39% identical to RasGRP1, RasGRP2 and RasGRP3, and the most conserved region in the four human RasGRPs is the GEF domain. Mast cells express H-Ras, K-Ras, M-Ras, Rab3A, Rab3B, Rab3D, Rap1, Rac1, Rac2 and Cdc42. While recombinant RasGRP4 and

RasGRP4 REGULATES MAST CELL DEVELOPMENT AND FUNCTION WP:CE34003: 48 hRasGRP4: 89

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HQWLSDSLSLITHFVNFYQETRNVEQ---REAVCRAVSFWIEKFPMHFDAQPQVCAQVVR 104 H W S L YQ Q R C V W P PQ R HSWVLPSADLAARLLTSYQKATGDTQELRRLLICHLVRYWLMRHPEVMHQDPQLEEVIGR 148

WP:CE34003: 105 L-KTIAEDINENIRNGLDVSALPSFAWLRA-VSVRNP-LAKQTAFSLSFVQASPSDISTS 161 T A N R D S L S P L K SL F hRasGRP4: 149 FWATVAREGNSAQRRLGDSSDLLSPGGPGPPLPMSSPGLGKKRKVSLLFDHLETGELAQH 208 WP:CE34003: 162 LSHIDYRVLSRISITELKQYVKDGHLRSCPMLERSISVFNNLSNWVQCMILNKTTPKERA 221 L R I L YV G R CP LE S N S WVQ M L P RA hRasGRP4: 209 LTYLEFRSFQAITPQDLRSYVLQGSVRGCPALEGSVGLSNSVSRWVQVMVLSCPGPLQRA 268 WP:CE34003: 222 EILVKFVHVAKHLRKINNFNTLMSVVGGITHSSVARLAKTYAVLSNDIKKELTQLTNLLS 281 L KF HVA L NFNTLM V GG HS RL A LS D K L LT LL hRasGRP4: 269 QVLDKFIHVAQRLHQLQNFNTLMAVTGGLCHSAISRLKDSHAHLSPDSTKALLELTELLA 328 WP:CE34003: 282 AQHNFCEYRKALGACNKKFRIPIIGVHLKDLVAINCSGANFEKTKCISSDKLVKLSKLLS 341 N YR C FR P GVHLKDLV KL L L hRasGRP4: 329 SHNNYARYRRTWAGC-AGFRLPVLGVHLKDLVSLHEAQPDRLPDGRLHLPKLNNLYLRL- 386 WP:CE34003: 342 NFLVFNQKGHNLPEMNMDLINTLKVSLDIRYNDDDIYELSLRREPKTFMNFEPS--RGLV 399 LV Q H N DL L SLD Y D IYELS REP PS hRasGRP4: 387 QELVALQGQHPPCSANEDLLHLLTLSLDLFYTEDEIYELSYAREPRCPKSLPPSPFNAPL 446 WP:CE34003: 400 FAEWASGVTVAPDNATVSKHISAMVDAVFKHYDHDRDGFISQEEFQLIAGNFPFIDAFVN 459 EWA GVT PD T H V V FKYD G ISQE F GNFPF hRasGRP4: 447 VVEWAPGVTPKPDRVTLGRHVEQLVESVFKNYDPEGRGTISQEDFERLSGNFPFACHGLH 506 WP:CE34003: 460 IDVDMD-GQISKDELKTYFMAANKNTKDLRRGFKHNFHETTFLTPTTCNHCNKLLWGILR 518 G S EL Y A L F H FHE TF PT C C LWG hRasGRP4: 507 PPPRQGRGSFSREELTGYLLRASAICSKLGLAFLHTFHEVTFRKPTFCDSCSGFLWGVTK 566 WP:CE34003: 519 QGFKCKDCGLAVHSCCKSNAVAECRRK 545 QG C CGL H C EC hRasGRP4: 567 QGYRCRECGLCCHKHCRDQVKVECKKR 593

FIG. 2. Comparison of the amino acid sequences of human RasGRP4 and the C. elegans protein WP:CE34003. Shown are the amino acid sequences of the major RasGRP4 isoform present in human mast cells, as well as WP:CE34003 which is the predicted translated product of the C. elegans F25B3.3 gene. The putative GEF, Ca2+-binding, and DAG/phorbol ester receptor domains in WP:CE34003 (residues 142–387, 428–444 and 493–542, respectively) and RasGRP4 (residues 189–432, 475–491 and 541–590, respectively) are highlighted in grey. The GVHLKDLV sequence that resides in the middle of the GEF domain of RasGRP4 is present in all mouse, rat and human RasGRPs. Based on the crystal structure of Sos-1, it is likely that this conserved 8-mer sequence is essential for the RasGRP4-mediated activation of Ras and other GTPases. The fact that the sequence is present in WP:CE34003 further supports the conclusion that the uncharacterized C. elegans protein is a GEF. Ca2+ binds to many proteins that possess a type-1 EF-hand motif of FX3DX9/10ED(or E)F. RasGRP4 is dominantly inhibited by Ca2+, and this signalling protein possesses a type-1 EF-hand motif downstream of the protein’s GEF domain. The presence of a similar domain in the corresponding region of WP:CE34003 suggests that this C. elegans protein is also regulated by Ca2+. The DAG-binding domain in protein kinase C enzymes and in all RasGRPs consists of ~50 amino acids and posses the motif of HX12CX2CX13/14CX2CX4 HX2CX7C. The presence of a similar domain in WP:CE34003 suggests that this domain binds DAG analogous to RasGRP4. The critical F, D, E, H and C amino acids in the DAG- and Ca2+binding domains of the two RasGRPs are highlighted. RasGRP4 and its other family members are tightly regulated at the gene and mRNA levels to restrict their expression in cells (Table 1). As of October 2004, >330 000 C. elegans-derived dbESTs have been deposited in GenBank. The finding that only four of these dbESTs originate from the F25B3.3 gene indicates that the expression of this putative RasGRP also is under exquisite control in C. elegans.

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A

Dark grey = H-Ras

Light grey = Mouse RasGRP4 (REM & CDC25 domains)

l

REM Domain l l

CDC25/GEF Domain

l

B

DAG-Binding Domain:

Aberrant Isoform with 5 additional residues (shaded region on right-hand loop above)

Normal Isoform

FIG. 3. Comparative 3D models of different regions in mouse RasGRP4. (A) Cartoon representation of the 3D model of the Ras exchanger motif (REM) and the CDC25/GEF domain (residues 34–445) (light grey) in mouse RasGRP4 bound to H-Ras (dark grey). The 412-mer sequence was modelled using the MODELLER computer program and the crystal structure of human Sos-1 (Protein Data Bank code 1BKD). The overall structure of this region is predicted to be very similar to that in human RasGRP4 (Yang et al 2002) despite the fact that 50 amino acids differ. The two residues predicted to be essential for its interaction with H-Ras are shown in ball and stick representation. (B) Cartoon representation of the 3D model of the DAG/phorbol ester-binding domain in mouse RasGRP (residues 541–597). Two splice variants of RasGRP4 have been cloned from BALB/c mBMMCs that differ in their DAG/phorbol esterbinding domains (Yang et al 2002). The 3D model of the normal isoform (right panel) resembles that of the primary isoform in human RasGRP4. The aberrant isoform (left panel) contains a fiveresidue insertion. The model predicts that the aberrant isoform also recognizes DAG, but this has not been shown experimentally. The DAG/phorbol ester-binding domains of the two isoforms were modelled based on the crystal structure of rat protein kinase Cg (Protein Data Bank code 1TBN).

RasGRP2 can catalyse the transfer of GTP to recombinant H-Ras efficiently, the latter GEF also can activate Rap1 (Clyde-Smith et al 2000). It therefore remains to be determined which Ras family member is the preferred target of RasGRP4 in mast cells.


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Although RasGRP4 lacks a membrane-spanning domain, a substantial portion of RasGRP4 resides on the cytosolic side of the mast cell’s plasma membrane (Li et al 2003a). These data suggest that an accessory factor or post-translational modification event regulates the translocation and retention of RasGRP4 in the inner leaflet of the mast cell’s plasma membrane. Signal pathways in mast cells are exquisitely dependent on the subcellular location and temporal expression of the specific cassette of intracellular proteins the cell uses to respond to the activating signal that occurs at its plasma membrane. KitL stimulation of mast cells via c-Kit induces phospholipase C enzymes to hydrolyse phosphatidylylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsP3). The binding of DAG to the C1 domains in downstream signalling proteins enables the resulting hydrophobic complexes to bind to membranes. The transient expression of DAG has been linked to the pronounced morphological changes that occur in the cytokine-activated cells. The model of the three-dimensional structure of human (Yang et al 2002) and mouse (Fig. 3) RasGRP4 predicted that residues 537–590 closely resemble the crystal structure of the C1 domain in protein kinase C. The phorbol ester/DAG-binding, C1 domain in protein kinase C is ~50 residues long and possesses the linear motif of HX12CX2CX13/14CX2CX4HX2CX7C, where H is His, C is Cys, and X is any other amino acid. As noted in Fig. 2, all of the critical His and Cys residues are present in RasGRP4, as well as in its C. elegans orthologue. The fact that phorbol-12-myristate 13-acetate (PMA) treatment of RasGRP4-expressing fibroblast transfectants results in dramatic morphological changes and substantial changes in actin organization confirmed that RasGRP4 is a phorbol ester receptor (Yang et al 2002). Subsequent deletion and site-directed mutagenesis/expression studies supported the conclusion that residues 537–590 in RasGRP4 comprise its PMA/DAGbinding site (Li et al 2003a). The accumulated data suggest that RasGRP4 requires DAG to translocate to the plasma membrane to encounter its target GTPase. How RasGRP4 is negatively regulated in mast cells remains to be determined experimentally. However, one potential negative control point is the catabolism of DAG after this signalling protein binds DAG and carries out its task inside the mast cell. In regard to this possibility, DAG kinase (DGK)a negatively regulates RasGRP1-dependent signalling in T cells by converting DAG to phosphatidic acid (Jones et al 2002). Nine mammalian DGKs have been cloned. As assessed by GeneChip analysis, cord blood human mast cells and the human mast cell line HMC-1 lack the transcripts that encode DGKs a, g, e or q. Rather, these immune cells express DGKs d and z. Whereas DGK z is expressed in a number of tissues and cell types, the observation that DGK d is more restricted raises the possibility that the latter enzyme is the DGK that preferentially dampens RasGRP4dependent signalling in activated mast cells. Ca2+ binds to many proteins that possess the EF-hand motif of FX3DX9/10ED(or E)F, where F is Phe, D is Asp, E is Glu, and X is any other amino acid. Each mam-

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malian and C. elegans RasGRP possesses at least one EF-hand motif C-terminal of its GEF domain (Fig. 2). Using an expression/site-directed mutagenesis approach, Ebinu et al (1998) demonstrated that RasGRP1 uses this EF-hand motif to bind Ca+2. RasGRP4 possesses a similar motif, and Ca+2 dominantly inhibits the ability of recombinant RasGRP4 to activate H-Ras even in the presence of a 15-fold molar excess of Mg2+ (Yang et al 2002). The accumulated data raise the possibility that RasGRP4 is also a Ca+2 sensor in activated mast cells and that RasGRP4-mediated signalling events are dampened when the cytosolic levels of Ca+2 are unusually high as occurs in FceRI-mediated responses. Role of the RasGRP family of signalling proteins in the development of T cells, B cells, platelets and mast cells While the primary function of the RasGRP protein WP:CE34003 in C. elegans has not been deduced, mouse and human T cells express RasGRP1 and targeted disruption of its gene in mice results in a marked diminution in the number of mature CD4+ and CD8+ T cells (Dower et al 2000). In support of the conclusion that RasGRP1 plays an essential role in the final stages of T cell development, overexpression of RasGRP1 in the thymus of the mouse leads to a substantial increase in the number of CD8+ T cells (Norment et al 2003). Although B cells express RasGRP1 (as well as RasGRP3), no obvious defects in B cells were observed in RasGRP1-null mice or in transgenic mice that over express RasGRP1. The subsequent discovery that signalling via the B cell antigen receptor (surface immunoglobulin) is diminished significantly in RasGRP3-deficient B cell lines (Oh-hora et al 2003) suggests that RasGRP3 plays a more prominent role in the final stages of B cell development. Megakaryocytes preferentially express RasGRP2, and mouse platelets that lack RasGRP2 are severely compromised in terms of their ability to bind to collagen and form thrombi (Crittenden et al 2004). Because of this GEF defect, RasGRP2-null mice are unable to clot quickly in a standard tail-bleed assay. RasGRP4 lacks the SH2 and SH3 domains that are present in Vav1–3. Based on these substantial structural differences, RasGRP4 and the Vav proteins must be functionally dissimilar in mast cells. In support of this conclusion, RasGRP4 does not reside in the nucleus (Li et al 2003a) as does Vav1. All mast cells express c-Kit and RasGRP4, and c-Kit is the only cytokine receptor that is absolutely essential for the development of tissue mast cells. Mast cells undergo substantial morphological changes when they bind to KitL-expressing mesenchymal cells, W/W v mice contain substantial numbers of MC-committed progenitors in their bone marrow, and mast cells that express multiple granule proteases can be generated from W/W v mice by culturing their bone marrow progenitors in the presence of IL3. Nevertheless, the tissues of W/W v mice normally contain very few granulated mast cells because of a genetic defect in the intracellular tyrosine kinase domain of c-Kit that regulates down-


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stream signalling pathways inside mature MCs and their progenitors. Despite these data, substantial numbers of mature mast cells develop in the skin of W/W v mice following topical exposure of the skin to a phorbol ester (Gordon & Galli 1990). The latter study raised the possibility that an undefined phorbol ester/DAG-dependent signalling protein downstream of c-Kit controls the mast cell’s final stages of development. The finding that RasGRP4-expressing fibroblasts undergo dramatic morphologic changes when exposed to phorbol myristate acetate (PMA) raised the possibility that RasGRP4 is a nonkinase-type PMA/DAG-dependent signalling protein that helps regulate the development and morphology of KitL-treated mast cells. Systemic mastocytosis and mast cell leukaemia are heterogeneous disorders that lead to the production of excessive numbers of immature mast cells. Many patients with mastocytosis possess point mutations in the cytoplasmic domain of c-Kit that cause their tissue mast cells to be in a heightened state of activation. This gain-infunction mutation was first identified in the HMC-1 cell line (Furitsu et al 1993) established by Butterfield and co-workers from a patient with a mast cell leukaemia (Butterfield et al 1988). Despite the fact that HMC-1 cells possess an activating mutation that should enhance cellular differentiation and granule maturation, these leukemia cells are less mature than mBMMCs. For example, they do not express prostaglandin (PG) D2, which is the predominant eicosanoid produced by KitLdependent cutaneous mast cells. To explain these inconsistencies, we concluded that HMC-1 cells probably have an additional defect downstream of c-Kit that dominantly prevents these cells from developing into more recognizable mast cells. Although we subsequently found that the RasGRP4 gene is transcribed in a subpopulation of HMC-1 cells, these cells produced only inactive isoforms of RasGRP4 due to an inability to remove introns 3 and 5 in the processed transcript. Whether or not this splicing abnormality is common in any patient group remains to be determined. Nevertheless, the identification of a tryptase-expressing human mast cell line that lacks functional RasGRP4 was a fortuitous finding in that it gave us the opportunity to identify the primary target genes that are regulated by the signalling protein in constitutively c-Kit-activated mast cells. HMC-1 cells continued to proliferate when they were induced to express the normal isoform of RasGRP4. While these data suggest that RasGRP4 is probably not essential for the viability and proliferation of mast cells and their progenitors, many of the transfectants developed electron-dense granules, dramatically increased their granule tryptase content, and began expressing small amounts of chymase and carboxypeptidase A3. However, the pathway that was most prominently regulated by RasGRP4 in the transfectants was the one that controls PGD2 biosynthesis (Li et al 2003b). All non-transformed rodent and human mast cells examined to date preferentially metabolize arachidonic acid via the cyclooxygenase pathway to PGD2. While it is known that c-Kit and MITF control PGD2 expression in mast cells by regulating the steady-state levels of the synthase that converts PGH2 to PGD2, the

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intermediate intracellular proteins in this signalling pathway had not been identified. RasGRP4-defective HMC-1 cells produce virtually no PGD2 (Macchia et al 1995) due to the absence of the terminal synthase used in its biosynthesis (Li et al 2003b). These findings allowed us to use HMC-1 cells, which are constitutively cKit-activated (Furitsu et al 1993), to further elucidate the intracellular signalling pathways that dominantly control PGD2 production in this cell line. GeneChip analysis of ~13 000 transcripts in RasGRP4- and RasGRP4+ HMC-1 cells revealed a >100fold difference in the levels of haematopoietic-type PGD2 synthase mRNA (Li et al 2003b). As assessed by SDS-PAGE immunoblot analysis, RasGRP4+ HMC-1 cells contained large amounts of PGD2 synthase protein. The transfectants also produced substantially more PGD2 eicosanoid than did RasGRP4- HMC-1 cells when both cell populations were exposed to a calcium ionophore. RBL-1 and 2H3 are related rat mast cell lines that constitutively express RasGRP4 (Li et al 2003a) and PGD2 (Westcott et al 1996). Because these cells are also transfectable, an RNA interference approach was used to confirm that RasGRP4 controls PGD2 synthase expression in MCs (Li et al 2003b). The accumulated data suggest that RasGRP4 controls the final stages of mast cell development, including the panel of protease and lipid mediators that this immune cell expresses. Pathological consequences of abnormal expression of a RasGRP Dysregulation of RasGRP expression can have profound consequences in mice. For example, forced expression of RasGRP1 (Ebinu et al 1998, Tognon et al 1998), RasGRP2 (Clyde-Smith et al 2000), RasGRP3 (Yamashita et al 2000) or RasGRP4 (Yang et al 2002) in cultured fibroblasts leads to decreased contact inhibition and actin reorganization, and increased proliferation when the transfectants are exposed to PMA. These findings raise the possibility that the presence of an unusually high level of a RasGRP in a cell can lead to its increased susceptibility to transformation. Whether or not a RasGRP plays a significant role in any proliferation disorder in humans remains to be determined. Nevertheless, the spontaneous insertion of a mouse leukaemia virus into the 5¢ flanking regions or introns of the RasGRP1 gene, the RasGRP2 gene, or at a chromosome 7A3-B1 site near where the RasGRP4 gene resides often leads to myeloid leukaemia, B cell lymphoma, and/or T cell lymphoma in mice (Li et al 1999, Suzuki et al 2002). As noted at the website http://genome2.ncifcrf.gov/RTCGD, 24 mouse leukaemias have already been identified in which the transforming virus inserted either in the 5¢ flanking region, intron 1 or intron 2 of the mouse RasGRP1 gene. Interestingly, most of the RasGRP4 dbESTs in GenBank’s database (Table 1) originated from acute myelogenous leukaemia cells or from clear-cell tumours of the kidney. A number of splice variants of mouse, rat, and human RasGRP4 have been identified (Li et al 2002, Li et al 2003a, Reuther et al 2002, Yang et al 2002). As


65

noted in HMC-1 cells and in C3H/HeJ mBMMCs, the failure to properly splice a RasGRP4 precursor transcript can have profound pathologic consequences. The other RasGRP genes also undergo differential splicing and an inbred mouse strain (designated as the lag mouse) was recently identified that spontaneously developed a lymphoproliferative autoimmune syndrome that resembles that found in patients with systemic lupus erythematosus (Layer et al 2003). The T cells in lag mice lack RasGRP1 protein due to an unexplained failure to remove intron 3 from its precursor transcript. The failure to remove intron 3 causes a premature translation– termination codon. As noted above, a similar situation occurs in the RasGRP4 transcript that is expressed in HMC-1 cells (Yang et al 2002). Because those RasGRP splice variants that have not removed intron 3 encode non-functional proteins, mast cells and T cells use an undefined post-transcriptional mechanism to control the intracellular levels of their functional RasGRP proteins. An exon 2/intron 2 splice-site mutation in the mouse mast cell protease 7 gene causes aberrant processing of this tryptase transcript in C57BL/6 mouse mast cells. Sequence analysis failed to reveal a comparable genetic abnormality in exon 3, intron 3 or exon 4 of the RasGRP4 gene in the HMC-1 cell line. Thus, the posttranscriptional defect that selectively hinders removal of intron 3 in the RasGRP4 gene in the HMC-1 cell line remains to be determined experimentally. Nevertheless, the unique features of this intron offer a clue as to what might be occurring. The maturation of all mammalian mRNAs occurs by two distinct splicing pathways that are dependent on the type of intron and its recognition by different small nuclear ribonucleoprotein particles. Sequence analysis of the 17 introns in the mouse and human RasGRP4 genes revealed that intron 3 is a rare U12-dependent intron (for review of U12 introns, see Patel & Steitz 2003). It has been estimated that only ~0.1% of the introns in the human genome are U12-dependent. The rest of the introns are U2-dependent, as are the other 16 introns in the RasGRP4 gene. It is interesting that all four RasGRP genes in the mouse and human genomes possess this rare U12-dependent intron. Because the splicing of an U12-dependent intron in a precursor transcript can be the rate-limiting step in gene expression (Patel et al 2002), the conserved U12-dependent intron that is located in all four RasGRP genes immediately upstream of the exons that encode the GEF domain in each protein probably helps regulate the levels of functional RasGRP transcripts in cells. Asthma is a complex polygenic disorder that is influenced additionally by a variety of environmental factors (e.g. antigen exposure). Many genes that are expressed in mast cells or encode products that influence mast cell development and/or function have been implicated in this pulmonary disorder. Airway functional studies revealed that A/J mice are intrinsically much more sensitive to methacholine than C3H/HeJ mice (De Sanctis et al 1999). In their gene-linkage study, De Sanctis and co-workers (De Sanctis et al 1999) noted that intrinsic airway reactivity to methacholine in backcrossed C3H/HeJ and A/J mice is dominantly influenced by a site

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on mouse chromosome 7A3-B1 near the D7Mit247 genetic marker. Because this site was not identified in backcrossed A/J and C57BL/6 mice (De Sanctis et al 1995), the C3H/HeJ mouse apparently differs from the A/J and C57BL/6 mouse strains in that it contains a quantitative trait locus on chromosome 7 that influences its intrinsic airway sensitivity to methacholine. Mast cells regulate allergic inflammation and play important immunoregulatory roles in the lung. Because the mouse RasGRP4 gene resides at chromosome 7A3-B1, the De Sanctis et al 1999 study raised the possibility that a defective isoform of RasGRP4 might be expressed in the mast cells of the hyporesponsive C3H/HeJ mouse. Analysis of the mBMMCs developed from C3H/HeJ mice revealed that the size of the predominant RasGRP4 transcript present in this mouse strain is slightly larger than the corresponding transcripts in A/J, BALB/c, and C57BL/6 mBMMCs (Li et al 2003a). Sequence analysis revealed that this RasGRP4 transcript lacks 22 nucleotides because of the preferential use of a cryptic splice-site in the middle of exon 15. This RNA-editing event results in a premature translation-termination codon. The resulting 578residue isoform lacks its DAG-binding domain; it also contains five different amino acids at its C-terminus. Fibroblast transfectants that express the normal, full-length RasGRP4 isoform that is present in BALB/c, C57BL/6 and A/J mBMMCs undergo dramatic morphologic changes when exposed to PMA (Yang et al 2002). This morphological feature did not occur in those transfectants that had been induced to express the truncated RasGRP4 isoform that is present in C3H/HeJ mBMMCs. While there could be another gene on mouse chromosome 7A3-B1 that also is defective in the C3H/HeJ mouse strain, the data raise the possibility that the unidentified gene noted in the De Sanctis et al (1999) study that impacts intrinsic airway reactivity in the C3H/HeJ mouse strain is the gene that encodes RasGRP4. Mouse chromosome 7A3-B1 corresponds to human chromosome 19q13.1, and a quantitative trait locus residing at or just downstream of the RasGRP4 gene has been linked to asthma and airway hyperresponsiveness in Caucasians (The Collaborative Study on the Genetics of Asthma 1997) and Hutterites (Ober et al 2000). An in-depth single nucleotide polymorphism (SNP) analysis of the human RasGRP4 gene has not yet been carried out. Nevertheless, sequence analysis of eight cDNAs arbitrarily isolated from a Clontech cDNA library (created using pooled RNA from the peripheral blood mononuclear cells of 550 individuals) resulted in the identification of 10 SNPs in the human RasGRP4 transcript that result in five amino acid differences in the translated products (Yang et al 2002). Three of these allelic differences result in non-conservative changes in the protein’s primary amino acid sequence. Interestingly, the variable amino acid at residue 335 is predicted to reside on the face of the protein that interacts with H-Ras. While non-functional isoforms of the signalling protein that are caused by differential splicing of the precursor transcript also have been identified in human blood leukocytes, it remains to be determined in future studies if any of these RasGRP4 iso-


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forms adversely affect patients with asthma, systemic mastocytosis, acute basophilic leukaemia, mast cell leukaemia or allergic disorders. Acknowledgements This work was supported in part by grant HL036110 and AI054950 from the National Institutes of Health and by grants from the National Health and Medical Research Council of Australia. References Butterfield JH, Weiler D, Dewald G, Gleich GJ 1988 Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res 12:345–355 Clyde-Smith J, Silins G, Gartside M et al 2000 Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. J Biol Chem 275:32260–32267 Crittenden JR, Bergmeier W, Zhang Y et al 2004 CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nat Med 10:982–986 De Sanctis GT, Merchant M, Beier DR et al 1995 Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice. Nat Genet 11:150–154 De Sanctis GT, Singer JB, Jiao A et al 1999 Quantitative trait locus mapping of airway responsiveness to chromosomes 6 and 7 in inbred mice. Am J Physiol 277:L1118–L1123 Dower NA, Stang SL, Bottorff DA et al 2000 RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol 1:317–321 Ebinu JO, Bottorff DA, Chan EY, Stang SL, Dunn RJ, Stone JC 1998 RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 280:1082–1086 Furitsu T, Tsujimura T, Tono T et al 1993 Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest 92:1736–1744 Gordon JR, Galli SJ 1990 Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/W v but Sl/Sl d genetically mast cell-deficient mice. Blood 75:1637–1645 Jones S, Vignais ML, Broach JR 1991 The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to Ras. Mol Cell Biol 11:2641–2646 Jones DR, Sanjuan MA, Stone JC, Merida I 2002 Expression of a catalytically inactive form of diacylglycerol kinase a induces sustained signaling through RasGRP. FASEB J 16:595–597 Kawasaki H, Springett GM, Toki S et al 1998 A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. Proc Natl Acad Sci USA 95:13278–13283 Kedra D, Seroussi E, Fransson I et al 1997 The germinal center kinase gene and a novel CDC25like gene are located in the vicinity of the PYGM gene on 11q13. Hum Genet 100:611–619 Layer K, Lin G, Nencioni A et al 2003 Autoimmunity as the consequence of a spontaneous mutation in RasGRP1. Immunity 19:243–255 Li J, Shen H, Himmel KL et al 1999 Leukemia disease genes: large-scale cloning and pathway predictions. Nat Genet 23:348–353 Li L, Yang Y, Stevens RL 2002 Cloning of rat Ras guanine nucleotide releasing protein 4, and evaluation of its expression in rat mast cells and their bone marrow progenitors. Mol Immunol 38:1283–1288 Li L, Yang Y, Wong GW, Stevens RL 2003a Mast cells in airway hyporesponsive C3H/HeJ mice express a unique isoform of the signaling protein RasGRP4 that is unresponsive to diacylglycerol and phorbol esters. J Immunol 171:390–397 Li L, Yang Y, Stevens RL 2003b RasGRP4 regulates the expression of prostaglandin D2 in human and rat mast cell lines. J Biol Chem 278:4725–4729

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Macchia L, Hamberg M, Kumlin M, Butterfield JH, Haeggstrom JZ 1995 Arachidonic acid metabolism in the human mast cell line HMC-1: 5-lipoxygenase gene expression and biosynthesis of thromboxane. Biochim Biophys Acta 1257:58–74 Nagase T, Ishikawa K, Suyama M et al 1998 Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res 5:355–364 Norment AM, Bogatzki LY, Klinger M, Ojala EW, Bevan MJ, Kay RJ 2003 Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes. J Immunol 170:1141–1149 Ober C, Tsalenko A, Parry R, Cox NJ 2000 A second-generation genomewide screen for asthmasusceptibility alleles in a founder population. Am J Hum Genet 67:1154–1162 Oh-hora M, Johmura S, Hashimoto A, Hikida M, Kurosaki T 2003 Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-g 2 to Ras in B cell receptor signaling. J Exp Med 198:1841–1851 Patel AA, Steitz JA 2003 Splicing double: insights from the second spliceosome. Nat Rev Mol Cell Biol 4:960–970 Patel AA, McCarthy M, Steitz JA 2002 The splicing of U12-type introns can be a rate-limiting step in gene expression. EMBO J 21:3804–3815 Reuther GW, Lambert QT, Rebhun JF, Caligiuri MA, Quilliam LA, Der CJ 2002 RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia. J Biol Chem 277:30508–30514 Song JS, Gomez J, Stancato LF, Rivera J 1996 Association of a p95 Vav-containing signaling complex with the FceRI gamma chain in the RBL-2H3 mast cell line: evidence for a constitutive in vivo association of Vav with Grb2, Raf-1, and ERK2 in an active complex. J Biol Chem 271:26962–26970 Song JS, Haleem-Smith H, Arudchandran R et al 1999 Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway. J Immunol 163:802–810 Suzuki T, Shen H, Akagi K et al 2002 New genes involved in cancer identified by retroviral tagging. Nat Genet 32:166–174 The Collaborative Study on the Genetics of Asthma 1997 A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nat Genet 15:389–392 Tognon CE, Kirk HE, Passmore LA, Whitehead IP, Der CJ, Kay RJ 1998 Regulation of RasGRP via a phorbol ester-responsive C1 domain. Mol Cell Biol 18:6995–7008 Turner H, Reif K, Rivera J, Cantrell DA 1995 Regulation of the adapter molecule Grb2 by FceRI in the mast cell line RBL-2H3. J Biol Chem 270:9500–9506 Westcott JY, Wenzel SE, Dreskin SC 1996 Arachidonate-induced eicosanoid synthesis in RBL2H3 cells: stimulation with antigen or A23187 induces prolonged activation of 5-lipoxygenase. Biochim Biophys Acta 1303:74–81 Yamashita S, Mochizuki N, Ohba Y et al 2000 CalDAG-GEFIII activation of Ras, R-Ras, and Rap1. J Biol Chem 275:25488–25493 Yang Y, Li L, Wong GW et al 2002 RasGRP4, a new mast cell-restricted Ras guanine nucleotide releasing protein with calcium- and diacylglycerol-binding motifs: identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. J Biol Chem 277:25756–25774

DISCUSSION Rivera: Have you been able to analyse the ability of mast cells developed from C3H/HeJ mice to degranulate?


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Stevens: We haven’t carried out any activation/degranulation experiments on RasGRP4 defective C3H/HeJ mouse bone marrow-derived mast cells (mBMMCs). While C3H/HeJ mBMMCs preferentially produce a RasGRP4 isoform that lacks its regulatory diacylglycerol/phorbol ester-binding domain, the mouse strain compensates for the defect by dramatically increasing the levels of total RasGRP4 (Li et al 2003). Because we anticipate that it is going to be difficult to interpret RasGRP4-dependent data from C3H/HeJ mice, we are attempting to knock out the mRasGRP4 gene in C57BL/6 mice using a homologous recombination approach. Metcalfe: When you looked for expression of RasGRP4 early on, how far back in cell lineage can you trace its expression? You say it is in mast cell progenitors, but does its expression go back to pluripotential stem cells? Related to this question you said you sorted nucleated cells. Does this mean that it is expressed in monocytes and macrophages? Stevens: RasGRP4 is expressed in all mature mouse, rat and human mast cells (Yang et al 2002, Li et al 2003). RasGRP4 is also expressed in an undefined population of non-granulated mononuclear cells in the blood. We suspect that the latter cells are the circulating progenitors that give rise to tissue mast cells. More than 6 million human dbESTs have been deposited in GenBank. Because only a few of these originate from the hRasGRP4 gene, RasGRP4 is a highly restricted signalling protein in vivo. It has been reported by others that macrophages and mast cells originate from a common progenitor (Valent et al 1989). As assessed immunohistochemically, RasGRP4 is not expressed in any tissue macrophage. RasGRP4 mRNA also has not been detected in those macrophage cell lines we examined. Based on these findings, the common progenitor that gives rise to mast cells and macrophages must cease expressing RasGRP4 when it is induced to become a macrophage. Metcalfe: So within the mononuclear cell population it is a mast cell progenitor but not a progenitor for monocytes and macrophages. Stevens: The RasGRP4-expressing mononuclear cells in blood have not been characterized. To identify and characterize these cells, we will place the Cre recombinase gene downstream of the translation-initiation site of the mRasGRP4 gene as we did to create an mMCP-6-promoter-Cre mouse (R. Adachi and R.L. Stevens, unpublished findings). If the resulting transgenic animals are viable, we should be able to mate them with the B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J mouse strain. This will result in the expression of GFP in the mRasGRP4-expressing progenitors in the mouse’s blood. Using a fluorescence-activated cell sorting (FACS) approach, we can then isolate and characterize mast cell-committed progenitors. For example, an Affymetrix GeneChip approach could be used to globally characterize transcript expression. Metcalfe: So you would say that as the cell differentiates into the mast cell lineage, that’s where it gets expressed.

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Stevens: RasGRP4 is expressed in every tissue mast cell and every mast cell line we have examined to date. In regard to your own work, in vitro-differentiated cord blood human mast cells express hRasGRP4. Do you accept Dr Valent’s conclusion that mast cells and macrophages originate from a common progenitor? Metcalfe: There are some data that mast cells and monocytes are more closely related in lineage compared with mast cells and lymphocytes (Kirshenbaum et al 1999). Stevens: RasGRP4 is not expressed in any normal tissue macrophages we have examined to date. For example, the mast cells that reside in the mouse’s peritoneal cavity contain abundant amounts of RasGRP4 protein. In contrast, the levels of RasGRP4 protein are below detection in mouse peritoneal macrophages. Metcalfe: It wouldn’t have to be expressed in the terminally differentiated cell, even if there is a common progenitor. Stevens: Peripheral blood CD4+ T cells, CD8+ T cells and CD19+ B cells lack RasGRP4 mRNA as assessed by RT-PCR analysis even though these lymphocytes express other RasGRP family members. Nevertheless, an undefined mononuclear cell in normal mouse and human blood contains substantial amounts of RasGRP4 mRNA. Whether these cells contain substantial amounts of RasGRP4 protein remains to be determined. Granulated mast cells are not present in normal mouse or human blood. Thus, the RasGRP4-expressing mononuclear cells are not mature mast cells. While we suspect that most of the RasGRP4-expressing cells are mast cell-committed progenitors, we have not ruled out the possibility that some of these cells are poorly granulated mouse basophils. Metcalfe: As mast cells develop, they begin to express the high-affinity IgE receptor. Most of the human mast cell-1(HMC1) cell lines don’t express the highaffinity IgE receptor. When you transfect these cells with RasGPR4, do they then express the high-affinity IgE receptor? Stevens: The RasGRP4 transfectants do not express the a chain of the highaffinity IgE receptor. Our data therefore imply that RasGRP4 does not regulate the expression of functional FceRI receptors. This is why we used calcium ionophore to activate HMC-1 cells and their RasGRP4-expressing transfectants. Razin: It’s likely that the knockout approach is not the right one. You probably will not find any mast cells. If RasGRP4 is only expressed in mast cells, what will you be able to do in the knockout mice? Stevens: W/W v and Sl/Sl d mice have been used by us and many others to evaluate the mast cell contribution in numerous in vivo studies. Unfortunately, these mast cell-deficient mice have low numbers of other c-Kit/stem cell factor-dependent cells (e.g. melanocytes and neutrophils). No one has generated a mouse that lacks just mast cells. Thus, it would be a major scientific advance if it was discovered that mRasGRP4-null mice have a selective loss of mast cells. Experiments could be carried out on these mice and their +/- littermates to evaluate the role of the mast cell in nearly any disorder (e.g. sepsis). In addition, mRasGRP4-expressing


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mBMMCs could be adoptively transferred into irradiated mRasGRP4-null mice to revert the phenotype back to that obtained in wild-type animals. However, it is possible that RasGRP4-null mice will resemble RasGRP1-null mice in that the transgenic animals will try to compensate for the loss of mature mast cells by increasing the number of mast cell-committed progenitors. The RasGRP4-defective HMC-1 mast cell line is viable. The HMC-1 cell line was isolated by Dr Butterfield’s group from a patient with a mast cell leukaemia (Butterfield et al 1988). Thus, it is also possible that targeted disruption of the RasGRP4 gene will result in an increased incidence of myeloid/mast cell leukaemias. Razin: You can think of something else. You could do a conditional knockout which would mean that you would be able to eliminate mast cells in vivo. What happens to the proliferation of mast cells when RasGRP4 is eliminated? Stevens: RasGRP4 has not been eliminated in a non-transformed mast cell to evaluate its impact on cell proliferation. Nevertheless, the RasGRP4-defective human mast cell line HMC-1 divides every 24 hours. Because the RasGRP4-expressing transfectants continue to proliferate, it is unlikely that this signalling protein is essential for the proliferation and viability of the mast cell progenitors that reside in the blood. RasGRP1 and RasGRP2 regulate the intermediate to final stages of T cell and platelet development, respectively. Our data suggest that RasGRP4 plays a similar role in controlling the intermediate to final stages of mast cell development. Razin: I had an observation a long time ago that when I treated bone marrowderived mast cells with phorbol myristate acetate (PMA), proliferation increased. Stevens: The mast cells in the jejunum of helminth-infected mice are proliferating and this population of mast cells express RasGRP4. While it remains to be determined whether RasGRP4 controls the viability and/or proliferation of jejunal mast cells or any population of cells, our data suggest that RasGRP4 controls the intermediate to final stages of mast cell development as I previously mentioned. In this regard, c-Kit ligand/stem cell factor induces mouse and human progenitors to increase storage of proteases in their granules. The cytokine also induces the cells to produce prostaglandin D2. The HMC-1 cell line was established from a patient with a mast cell leukaemia. Dr Kitamura’s group then showed that HMC-1 cells possess an activating mutation in the c-Kit gene (Furitsu et al 1993). The fact that the HMC-1 cells do not express prostaglandin D2 and chymase and express only small amounts of b tryptase raised the possibility that these transformed cells have a secondary defect downstream of c-Kit that controls protease and arachidonic acid expression but not cell proliferation and viability. Our data suggest that this is indeed the case and that the secondary defect is in RasGRP4 expression. Rivera: Does the RBL-2H3 cell line express an alternatively spliced form of RasGRP4? Stevens: Normal and abnormal rRasGRP4 transcripts are present in the rat basophilic leukaemia (RBL)-2H3 cell line but we have not shown that the abnormal transcripts are turned into abnormal protein. An aberrant RasGRP4 isoform

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could function as a dominant-negative as occurs with defective MITF in mi/mi mice. Alternately, it is possible that an aberrant RasGRP4 isoform does not negatively impact a mast cell or its progenitor as long as the cell contains a small amount of the normal isoform. The discovery that the HMC-1 cell line we received from Dr Butterfield’s group produces only defective RasGRP4 isoforms was fortunate because it allowed us to use a rescue approach to evaluate the consequences of RasGRP4 expression on gene and protein expression in a HMC cell line. Rivera: The diacylglycerol (DAG) binding site of RasGRP4 is of interest. Is this site important for targeting or for activity? Stevens: The levels of DAG are transiently increased in mast cells that have been activated via their c-Kit receptors. Normally, c-Kit-defective W/W v mice contain very few mast cells in their skin. Nevertheless, Gordon & Galli (1990) noted a marked increase in the number of granulated mast cell in the skin of phorbol estertreated W/W v mice. Phorbol esters are oncogenic because they recognize the DAG-binding sites in protein kinase C enzymes. The Gordon and Galli study raised the possibility of a signalling protein inside mast cells downstream of c-Kit that is regulated by phorbol esters and DAG. Prior to our 2002 RasGRP4 study, the only identified DAG-dependent proteins in mast cells were protein kinase C enzymes. We now know that RasGRP4 is also regulated by DAG. In regard to your location question, RasGRP4 lacks a membrane-spanning domain. Nevertheless, when we carried out an organelle-fraction study of lysed mBMMCs, ~50% of the immunoreactive RasGRP4 in the samples was recovered in the membrane fraction. Our immunogold studies of the mast cells in the V3 mastocytosis mouse also revealed that a substantial amount of RasGRP4 resides in the inner leaflet of the cell’s plasma membrane. These accumulated data suggest that RasGRP4 inserts in the plasma membrane when it binds a hydrophobic cofactor. The DAG/phorbol ester-binding domain in protein kinase C enzymes possesses the 50-mer sequence of HC12CX2CX13/14CX2CX4HX2CX7C where H is His, C is Cys and X is any other amino acid. Mouse, rat, and human RasGRP4 have this sequence in their C-terminal domains. Interestingly, the RasGRP-like protein F25B3.3 in C. elegans also has this sequence. The conservation of the DAG-binding motif for millions of years of evolution is strong circumstantial evidence of its importance. Rivera: Using your recombinant protein do you get activity in the absence of DAG? Stevens: Recombinant mouse and human RasGRP4 can activate recombinant Ras in vitro in the absence of DAG. A similar situation occurs for recombinant RasGRP1, RasGRP2 and RasGRP3. Most investigators in the signal transduction field believe that the four RasGRPs translocate from the cytosol to the inner leaflet of the plasma membrane when DAG is generated or when the cells encounter a phorbol ester.


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Rao: Where is the RasGRP-like protein expressed in C. elegans? Stevens: Our preliminary data suggest that the RasGRP-like protein F25B3.3 is preferentially expressed in the worm’s neurons. Rao: Is there only one RasGRP in C. elegans? Stevens: There is only one RasGRP-like gene in C. elegans. Very little is known about this gene. Nevertheless, the placement of the green fluorescent protein (GFP) gene immediately downstream of the RasGRP-like F25B3.3 gene results in GFP expression in the worm’s neurons. The C. elegans consortium is presently attempting to knockout every gene using either RNAi or gene-trap approaches. My co-worker Dr Barstead has knocked out the F25B3.3 gene and we are presently evaluating the worm’s phenotype. The four human RasGRPs are preferentially expressed in haematopoietic cells. Thus, why the F25B3.3 gene seems to be preferentially expressed in the neurons of C. elegans remains to be determined. One possibility is that F25B3.3 acts downstream of a neuronal receptor that is used by the worm to recognize microbial pathogens. Rao: If I understood your presentation, mast cells express RasGRP4 but not RasGRP1, RasGRP2 or RasGRP3. Stevens: That’s true for all mouse, rat, and human mast cells that we have examined. Rao: Have you tried complementing HMC-1 cells with either RasGRP1, RasGRP2 or RasGRP3? Stevens: RasGRP1-null mice have defects in T cell development (Dower et al 2000), whereas platelet development is defective in RasGRP2-null mice (Crittenden et al 2004). It also has been concluded that RasGRP3 regulates B cell development (Oh-hora et al 2003). It therefore is already known that the varied RasGRP family members are functionally distinct in haematopoietic cells. RasGRP4 is only ~50% identical to its closest family member. We did not carry out RasGRP1, RasGRP2 or RasGRP3 complementation experiments because we concluded it unlikely that another family member would be functional in mast cells. Nevertheless, I agree that the experiment should be carried out. We also should see if RasGRP4 can rescue the defect in T cell development in RasGRP1-null mice and the defect in platelet development in RasGRP2-null mice. Rao: Why do you predict that RasGRP4-null mice might tend to develop leukaemias? Stevens: The RasGRP area of investigation is relatively new, and most of what is known comes from studies on RasGRP1. Very few single-positive T cells are found in RasGRP1-null mice even though these transgenic mice contain normal numbers of CD4+/CD8+ T cells (Dower et al 2000). These data have led to the conclusion that RasGRP1 regulates the intermediate to final stages of T cell development. If a RasGRP1-null mouse is unable to produce normal numbers of mature T cells, it often increases the number of T cell-committed progenitors to try to compensate

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for the defect. This situation occurs in the lag mouse which possesses a defect in RasGRP1 expression due to an unexplained failure to remove intron 3 from its precursor transcript (Layer et al 2003). Dr Copeland’s group used a retroviral insertional mutagenesis approach to identify leukaemia susceptibility genes in mice (Suzuki et al 2002). That genome-wide hunt resulted in the identification of the RasGRP1 and RasGRP2 genes. Interestingly, a leukaemia-promoting site also was identified on mouse chromosome 7 near where the RasGRP4 gene resides. Nevertheless, no one has shown an association with any RasGRP gene and human leukaemia. Ono: I’d like to return to mast cells. Your initial slide showed c-Kit, and asked the question about the nature of the intermediate signalling molecule. The postulated downstream target was MITF. Do your data support RasGRP4 being upstream of MITF? In your gene chip analysis the gene that increases in the transfectant is prostaglandin D2 synthase. What is known about how this gene is regulated? Is it very likely to be RasGRP4 dependent? Stevens: Dr Austen and his co-workers reported that the expression of prostaglandin D2 synthase (PGDS) in mBMMCs is exquisitely regulated by a c-Kitdependent signalling pathway (Murakami et al 1995). Morii & Oboki (2004) recently reported that PGDS expression in mBMMCs is also controlled by MITF. RasGRP4expressing HMC-1 cells dramatically increase their PGDS expression, whereas RBL-2H3 cells that have been exposed to a RasGRP4-specific RNAi dramatically decrease their PGDS expression (Li et al 2003). The accumulated data suggest that RasGRP4 acts downstream of c-Kit and upstream of MITF. Possibly Dr Austen would like to comment on his c-Kit/Kit ligand studies of mBMMCs? Austen: IL3 and KitL/SCF have different effects on eicosanoid generating enzymes. KitL up-regulates PGD2 generation and IL3 up-regulates the cysteinyl leukotriene (cysLT) generating pathway. Stevens: Drs Morii and Oboki confirm in their study that a c-Kit ligand/stem cell factor (SCF)-dependent pathway regulates PGDS expression in mast cells. Using mast cells that are defective in MITF, they also showed that MITF is needed for PGDS expression. These investigators already had reported that MITF acts downstream of c-Kit in terms of controlling granule protease expression. Austen: One of the reasons we thought PGD2 was interesting is that every mast cell we looked at was a good source of this, irrespective of whether it could generate cysteinyl leukotrienes (cysLTs). In fact, it wasn’t possible to elicit slow reacting substance (SRS-A/cysLTs) from rat peritoneal mast cells, but it was easy to generate PGD2. Since the mast cell requires KitL to be present, it begins to make sense that virtually every mast cell is capable of making PGD2, whereas its ability to make cysLTs depends on it getting signals from additional cytokines. Ono: Do you interpret the fact that there wasn’t such an effect on protease expression to mean that regulation of protease genes is much more complex?


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Stevens: We focused our initiation attention on prostaglandin D2 synthase because the regulation of protease expression in mast cells must be much more complex in order to explain why RasGRP4-expressing mast cells can store different combinations of proteases in their secretory granules. While it has been reported that MITF is needed for the transcription of the mMCP-6 gene (Morii et al 1996) and probably other protease genes, we found that a post-transcriptional mechanism dominantly controls the expression of many proteases in mast cells (Xia et al 1996). Complicating the situation is the finding that mast cells express varied combinations of at least 16 neutral proteases. Mast cells express only one prostaglandin D2 synthase. Thus, it is much easier to understand how RasGRP4 controls the expression of this gene in mast cells. Our model is that RasGRP4 acts downstream of c-kit and upstream of MITF, but this remains to be proven experimentally. IL3developed mBMMCs metabolize arachidonic acid predominantly to leukotriene C4 rather than prostaglandin D2. RasGRP4-defective HMC-1 cells produce substantial amounts of leukotriene C4. Based on these data, it is unlikely that RasGRP4 is coupled to the IL3 receptor. Koyasu: I have a similar question. When you connect c-Kit with RasGRP4, your suggestion is that DAG is important in the activation. At the same time, if c-Kit connects with RasGRP4 but not IL3, there must be some other molecule, which might activate RasGRP4 with the aid of DAG. What is known about this? You mentioned the REM domain and this seems to have a specific sequence. Do you know anything about the other pathways? Stevens: We haven’t carried out an in-depth site-directed mutagenesis study to evaluate the importance of specific residues in the REM and other domains of RasGRP4. Nevertheless, in response to Dr Rivera’s question, we discovered that recombinant human and mouse RasGRP4 can activate recombinant Ras and other small GTP-binding proteins in the absence of DAG, at least in vitro. We presently don’t know which Ras family member is the preferred target of RasGRP4 inside a living mast cell. Although DAG does not appear to regulate the protein’s activity in vitro, this lipid probably plays a critical role inside the mast cell by targeting RasGRP4 to the appropriate membrane site where it can come in contact with the relevant Ras family member. Koyasu: Targeting to the membrane is one factor, but the other factor should come from c-Kit. Stevens: Receptor-mediated tyrosine phosphorylation of Vav1 controls the activity of this guanine nucleotide exchange (GNE) in mast cells and other cell types (Crespo et al 1997). It therefore is possible that activation of mast cells via c-Kit results in phosphorylation of RasGRP4 which, in turn, alters its activity and/ or specificity. Nevertheless, our studies and that of others carried out on RasGRP1, RasGRP2 and RasGRP3 suggest that the key cofactor that regulates this family of signalling proteins is DAG. Interestingly, DAG is generated when c-Kit

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ligand/SCF binds to c-Kit/CD117 on the surfaces of a mast cell (Koike et al 1993). Koyasu: Can another protein trigger the activation of RasGRP4? What are RasGRP4’s binding partners inside the mast cell. Stevens: RasGRP4 lacks the SH2 and SH3 domains that the Vav family of GNE factors use to bind to adaptor molecules inside mast cells. Nevertheless, it remains to be determined whether RasGRP4 binds to a specific adaptor protein. Koyasu: When you transfect 3T3 fibroblasts with RasGRP4, do the transfectants undergo morphological changes. Can you also do this with the other RasGRPs? Stevens: Yes. The fibroblast transfectants undergo dramatic morphologic changes when they are subsequently exposed to 10 nM phorbol ester (Yang et al 2002). A similar situation occurs for those fibroblast transfectants that express RasGRP1, RasGRP2, or RasGRP3. Koyasu: So this phenomenon is common to all RasGRP molecules? Stevens: Yes. Fibroblasts do not express RasGRP1, RasGRP2, RasGRP3 or RasGRP4. The transfection data imply that if a RasGRP is aberrantly expressed in the wrong cell type, the resulting cell probably will become more susceptible to a phorbol ester and thereby more prone to undergo a transformation event. MacDonald: You showed very nicely that it was not in unstimulated CD4+CD8+ cells. When you stimulated with lectin, this down-regulated it. But what I thought I saw on your slide was the appearance of a faint band in the CD4 cells? Is this true? Stevens: RasGRP4 is not an abundant transcript in CD4+ T cells before or after lectin treatment.

References Butterfield JH, Weiler D, Dewald G, Gleich GJ 1988 Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res 12:345–355 Crespo P, Schuebel KE, Ostrom AA, Gutkind JS, Bustelo XR 1997 Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385:169–172 Crittenden JR, Bergmeier W, Zhang Y et al 2004 CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nat Med 10:982–986 Dower NA, Stang SL, Bottorff DA et al 2000 RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol 1:317–321 Furitsu T, Tsujimura T, Tono T et al 1993 Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest 92:1736–1744 Gordon JR, Galli SJ 1990 Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/W v but Sl/Sl d genetically mast cell-deficient mice. Blood 75:1637–1645 Kirshenbaum AS, Goff JP, Semere T, Scott LM, Metcalfe DD 1999 Demonstration that human mast cells arise from a bipotential progenitor cell population that is CD34+, c-kit+, and expresses aminopeptidase N (CD13). Blood 94:2333–2342


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Koike T, Hirai K, Morita Y, Nozawa Y 1993 Stem cell factor-induced signal transduction in rat mast cells. Activation of phospholipase D but not phosphoinositide-specific phospholipase C in c-kit receptor stimulation. J Immunol 151:359–366 Layer K, Lin G, Nencioni A et al 2003 Autoimmunity as the consequence of a spontaneous mutation in RasGRP1. Immunity 19:243–255 Li L, Yang Y, Wong GW, Stevens RL 2003 Mast cells in airway hyporesponsive C3H/HeJ mice express a unique isoform of the signaling protein RasGRP4 that is unresponsive to diacylglycerol and phorbol esters. J Immunol 171:390–397 Morii E, Oboki K 2004 MITF is necessary for generation of prostaglandin D2 in mouse mast cells. J Biol Chem 279:48923– 48929 Morii E, Tsujimura T, Jippo T et al 1996 Regulation of mouse mast cell protease 6 gene expression by transcription factor encoded by the mi locus. Blood 88:2488–2494 Murakami M, Matsumoto R, Urade Y, Austen KF, Arm JP 1995 c-kit ligand mediates increased expression of cytosolic phospholipase A2, prostaglandin endoperoxide synthase-1, and hematopoietic prostaglandin D2 synthase and increased IgE-dependent prostaglandin D2 generation in immature mouse mast cells. J Biol Chem 270:3239–3246 Oh-hora M, Johmura S, Hashimoto A, Hikida M, Kurosaki T 2003 Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-g 2 to Ras in B cell receptor signaling. J Exp Med 198:1841–1851 Suzuki T, Shen H, Akagi K et al 2002 New genes involved in cancer identified by retroviral tagging. Nat Genet 32:166–174 Valent P, Ashman LK, Hinterberger W et al 1989 Mast cell typing: demonstration of a distinct hematopoietic cell type and evidence for immunophenotypic relationship to mononuclear phagocytes. Blood 73:1778–1785 Xia Z, Ghildyal N, Austen KF, Stevens RL 1996 Post-transcriptional regulation of chymase expression in mast cells. A cytokine-dependent mechanism for controlling the expression of granule neutral proteases of hematopoietic cells. J Biol Chem 271:8747–8753 Yang Y, Li L, Wong GW et al 2002 RasGRP4, a new mast cell-restricted Ras guanine nucleotide releasing protein with calcium- and diacylglycerol-binding motifs: identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. J Biol Chem 277:25756–25774

Regulation of mast cell secretory response to the type I Fce receptor: inhibitory elements and desensitization J. Abramson, E. A. Barbu and I. Pecht1 Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100, Israel

Abstract. While the current understanding of the stimulus–response coupling networks triggered by the multi-chain immune-recognition receptors (MIRRs) has markedly advanced, knowledge of its control mechanisms is only emerging. Regulation of the secretory response of mast cells to the stimulus provided by the type I Fce receptor (FceRI) is our topic of interest. Several mast cell membrane receptors capable of inhibiting both immediate and late responses have so far been identified. However, their ligands and mechanism(s) of operation are only partly known. Moreover, desensitization of mast cells’ response to the FceRI, a well-known and widespread control process of many neural or hormone receptors, is hardly understood in this case. In this brief report we describe results of recent experiments in which we studied both of these aspects of mast cells’ response to the FceRI stimulus by an inhibitory receptor MAFA, as well as those where we have established that these cells are susceptible to physiological modes of FceRI desensitization caused by prolonged exposure to sub-threshold concentrations of FceRI clustering agents. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 78–94

The overwhelming power of the immune system makes it essential to maintain it under an effective control and avoid falsely oriented or excessive reactivity. This is particularly important regarding the activation signals produced by members of the multi-chain immune recognition receptors (MIRR) family (FcR, TCR, BCR) responsible for exerting the adaptive responses of the immune system. One of the most effective inhibitory mechanisms regulating the MIRR-induced positive signalling involves members of another group of related membrane components termed the inhibitory receptor superfamily. All these receptors contain within their intracellular domains one or more copies of an immunoreceptor tyrosine-based inhibitory motif (ITIM), which is central for their inhibitory capacities (Cambier 1995, Reth 1989). In this report we briefly describe recent results of the molecular analysis of 1

This paper was presented at the symposium by I. Pecht to whom correspondence should be addressed. 78

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mechanisms by which an inhibitory receptor MAFA interferes with the type I Fce receptor (FceRI)-induced secretion of de novo synthesized mediators. A closely related issue of regulation is whether the MIRR’s signalling, particularly that of the FceRI could be attenuated by desensitization (i.e. by applying excessive or prolonged exposure to the ligand) in a comparable manner to that already known for hormonal or neural receptors. Essentially all the results reported so far on FceRI desensitization were based on experimental protocols that employed FceRI-mediated stimulation under response-non-permissive conditions (e.g. by limiting extracellular calcium ion concentrations) (Nakamura & MacGlashan 1994, MacGlashan & Lichtenstein 1987, Ishizaka et al 1985, Sobotka et al 1979, Weetall et al 1993). Therefore, we have recently investigated whether and to what extent desensitization of FceRI-induced secretory response is susceptible to more physiological conditions (Barbu et al 2002). The mast cell functional antigen (MAFA) Regulation of response to the FceRI stimulus by an inhibitory receptor was pursued by studies of a membranal glycoprotein discovered by us almost two decades ago and named mast cell function-associated antigen (MAFA). This protein was first identified on the surface of rat mast cells of the rat basophilic leukaemia (RBL)2H3 line on the basis of its capacity to inhibit these cells’ response to the FceRI induced secretion (Ortega & Pecht 1988). Expression cloning of MAFA cDNA has later shown that the molecule contains a single open reading frame encoding a 188 amino acid long sequence. The deduced 34 amino acid long intracellular domain contains a conserved ITIM sequence (SIYSTL) classifying MAFA as a member of the inhibitory receptors family (Guthmann et al 1995). However, unlike the majority of other ITIM-containing receptors, MAFA inhibits the activating stimulus without the requirement for its co-clustering with an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor. MAFA clustering alone, shortly prior to that of the FceRI, suffices for suppressing the secretory responses. MAFA’s 114 amino acid extracellular domain displays a marked homology with the carbohydrate recognition domain (CRD) of several members of the calcium dependent (C-type) lectins superfamily, such as CD23, CD69, CD72 and the natural killer (NK) cell receptors (Guthmann et al 1995). Significantly, MAFA homologues were more recently found to be expressed by humans and mice (Blaser et al 1998, Butcher et al 1998). However, these homologues were shown to display a rather different cellular expression pattern, i.e. they are expressed also or only by NK- and T-cells, where they most probably have different functions. Investigating the mechanism of MAFA’s inhibitory action, we have shown that upon its aggregation (by a specific mAb, G63) a rapid phosphorylation of its ITIM tyrosyl (as well as seryl) residue(s) is taking place. The tyrosyl phosphorylated ITIM

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provides a docking site for the SH2 domain-containing protein tyrosine phosphatase (SHP-2) and SH2 domain-containing inositol-polyphosphate 5-phosphatase (SHIP) (Xu et al 2001). SHP-2 was shown to interfere with the most proximal events to the activating FceRI, by dephosphorylating both Syk and LAT (linker for activation of T cells), thereby also suppressing the downstream coupling processes (Xu & Pecht 2001). However the SHP-2-mediated inhibition was found to represent only one (and most likely the minor) pathway, which mediates MAFA’s inhibitory activity. Indeed, results of experiments examining the role of SHIP have established that this enzyme plays the major role in MAFA mediated inhibition of mast cell degranulation (Xu et al 2001). Resolving how MAFA suppresses the FceRI-induced de novo synthesis and secretion of cytokines, chemokines and leukotrienes has been the focus of the next research phase. We have first used the RT-PCR and tested the mRNA levels of several cytokines or chemokines produced in response to FceRI or MAFA+FceRI clustering. As expected, the FceRI stimulation of RBL-2H3 cells leads to a dramatic increase in the mRNA levels of the examined cytokines. However, this increase was in most cases inhibited in cells where MAFA was clustered prior to the FceRI stimulation (Fig. 1). Interestingly, several cytokines (interleukin [IL]3, IL5) were unaffected by MAFA clustering (Fig. 1), suggesting that MAFA modulates the process of cytokine de novo synthesis in a rather selective manner. To resolve the molecular mechanism which MAFA employs for such a selective inhibition process, we next focused on molecules which are known to directly couple the FceRI stimulus to the gene transcription processes. These include primarily the mitogen-activated protein kinase (MAPK) family members such as Erk (extracellular signal-regulated kinases), p38 and Jnk (c-Jun N-terminal kinase) as well as another protein S/T kinase, Akt. Results of these studies demonstrated that MAFA interferes with FceRI-induced activation of both Erk-1/2 and p38 MAP kinases, while the activation of JNK and Akt was unaffected by MAFA clustering. These data therefore suggest that the previously observed selective inhibition of cytokine de novo synthesis is due to an interference of MAFA with the respective MAPK signalling pathways. In addition, two inhibitory adaptor molecules, Dok-1 and Dok-2, were found to play a central role in this regulatory process. They were both found to undergo phosphorylation of their tyrosine residues upon MAFA clustering with the concomitant increase in their binding to RasGAP. This binding is apparently essential for the MAFA-mediated down-regulation of RasGTP levels culminating in suppression of Erk activity and the subsequent reduction in the de novo synthesis and secretion of the cytokines and leukotrienes. Moreover, we have shown that both Dok proteins also undergo tyrosine phosphorylation upon FceRI clustering, suggesting that they are part of the FceRI autoregulatory apparatus. In order to gain more insights into Doks’ role in the FceRI signalling, RBL-2H3 cells with stable overexpression of Dok-1 were constructed.

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FIG. 1. MAFA clustering suppresses the FceRI-induced de novo synthesis of cytokines’ mRNA in a selective manner. Adherent RBL-2H3 cells (1 ¥ 107) were first reacted for 2 hours with the DNP specific A2IgE (2mg/10 ml). Cells were then washed and either left untreated as control or stimulated by FceRI-IgE aggregation (30 ng/ml BSA-DNP11) or MAFA clustered (100 nM mAb G63) + FceRI-IgE aggregation (30 ng/ml of BSA-DNP11) for 3 hours. Cells were then lysed and the total RNA was isolated using RNeasy mini kit (Qiagen) according to manufacturer’s instructions and levels of respective cytokines were measured by RT-PCR.

Studies using these cell variants clearly demonstrated that Dok-1 overexpression significantly suppresses the FceRI mediated Ras and Erk-1/2 activation, and concomitantly the de novo synthesis of tumour necrosis factor (TNF)a and leukotriene C4 (LTC4) (Fig. 2). These findings led us to propose that Dok-1 indeed functions as a built-in autoregulatory element, keeping in check the de novo synthesis of proinflammatory mediators induced upon FceRI aggregation (Abramson et al 2003). Desensitization of FceRI-induced degranulation by prolonged exposure to sub-threshold receptor clustering We investigated the possibility of inducing desensitization of mucosal-type mast cells’ secretory response to the FceRI stimulus by subjecting cells of the RBL-2H3 line to FceRI clustering agents at concentrations lower than those required for inducing secretion (sub-threshold) for prolonged periods of time (12–14 h). The secretory dose-response to two types of FceRI aggregating agents (IgE- and its specific antigen or IgE-specific mAb 95.3) was assayed and used to determine both the threshold and optimal concentrations required for inducing secretion of granule-stored mediators (monitored by the activity of the secreted bhexosaminidase). These were determined following loading of the cells with different concentrations (0.05, 0.5 and 5 nM) of the monomeric fraction of the DNP specific, monoclonal A2IgE. As expected, the antigen concentration threshold

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FIG. 2. Erk-1/2 activation, TNF-a and LTC4 secretion are all inhibited in RBL-2H3 cells overexpressing Dok-1. (A) Parental RBL-2H3 cells (parent.) or their mutants overexpressing either wild type (wt-) or mutated (mt-) Dok-1 were antigen-stimulated for three minutes. Equal amounts of protein samples were taken from cell lysates, separated by SDS-PAGE and electro-transferred onto nitrocellulose membranes. Erk-1/2 activation and the levels of proteins were monitored by sequential immunoblotting with antibodies specific for phosphorylated Erk-1/2 (pErk-1/2), total Erk-1/2 (Erk-1/2) or for the HA-tag (HA). (B) RBL-2H3 cells (white) and their wt-Dok-1 variants (black) were plated into 96 well plates (1 ¥ 105 cells/100 ml/well) and reacted for 2 hours with A2IgE (2 mg/10 ml). Cells were then washed and incubated with indicated concentrations of antigen for 24 h (B) or 30 minutes (C). Supernatants of the above cells were then assayed for TNFa levels or (C) LTC4 by ELISA according to the manufacturer’s instructions.

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values (rather than the optimal ones) exhibited a marked dependence on the employed IgE concentration. To establish the induction of desensitization and determine its dependence on the concentration of desensitizing antigen, three different concentrations were used (2.6, 4.4 and 5.6 ng/ml), all within the range previously shown to be sub-threshold and shown to cause a significant decrease in degranulation. Non-desensitized control cells and those exposed to desensitization were then stimulated for 30 min with a wide range of Ag concentrations (0.1–3000 ng/ml) and their secretory response was determined. The extent of desensitization was found to depend on the employed desensitizing antigen concentrations. As illustrated in Fig. 3A and 3B respectively, desensitizing with 2.6 ng/ml antigen caused a 65% decrease in secretion, whereas 4.4 ng/ml caused an 80% decrease. Treatment with the highest desensitizing antigen concentration (5.6 ng/ml) was found to cause the largest decrease in response; the secretory response was desensitized up to 90% at the optimal stimulation range and did not decline below 40% for the rest of the stimulation concentrations (Fig. 3C). Desensitization has also been investigated by using a different FceRI clustering agent, namely the IgE specific mAb 95.3. RBL-2H3 cells previously incubated with the monomeric mAb A2IgE (0.05 or 0.5 nM) were exposed to a 12 h desensitization period with two mAb 95.3 concentrations (0.3 or 0.4 nM; both previously determined as sub-threshold) or to medium only (as a control). Desensitized and nondesensitized cells were afterwards stimulated for 30 min with a range of mAb 95.3 concentrations (0.03–100 nM) and degranulation determined. Also in this case, the decrease in the secretory response caused by desensitization was found to correlate with the increase of the desensitizing agent concentrations. At both IgE concentrations, desensitization caused by treatment with 0.3 nM of mAb 95.3 reached 30% within the optimal stimulation range, while treatment with 0.4 nM of the clustering agent decreased the secretory response by 90% (data not shown). Interestingly, no marked dependence of the desensitization on the employed IgE concentrations could be resolved. The reason for this behaviour may probably be that mAb 95.3, being an IgG isotype is simultaneously binding to both its receptor—the low affinity receptor for IgG, Fcg RIIb (an ITIM-containing receptor also expressed by the RBL-2H3 cells) and to the IgE-FceRI complexes. The coclustering of Fcg RIIb with the activating FceRI counterpart is known to inhibit the FceRI-mediated secretion (Daeron et al 1995). Thus, the induced decrease in the secretory response observed when the mAb 95.3 was used as desensitizer might partially be caused by the inhibitory effect of the Fcg RIIb. Taken together, the above results clearly illustrate the possibility of inducing desensitization of mucosal mast cells, as illustrated by the rat RBL-2H3 line, by an extended exposure to sub-threshold FceRI aggregation under essentially physiological conditions.

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FIG. 3. Antigen concentration dependence of the degranulation of desensitized cells. RBL2H3 cells plated in 96 well plates (1 ¥ 105 cells/100 ml/well) were allowed to adhere and then reacted for 2 hours with 0.05 nM of the monomeric A2IgE. For desensitization cells were first washed with medium and incubated for 12–14 hours in medium containing one of the following desensitizing concentrations of antigen: 2.6 (A), 4.4 (B) or 5.6 (C) ng/ml. Control cells were maintained for the same time period in medium only. Afterwards, cells were washed and stimulated for 30 min. with the shown antigen concentrations. Degranulation of non-desensitized and desensitized cells was determined and presented as percentage of the cells’ total enzyme content (determined after lysis of control cells with 1% Triton). The decrease in secretion due to desensitization is presented as (%) difference between the desensitized response and the non-desensitized one (right ordinate). Data are from one experiment out of three and points represent average secretion of quadruplicates. Error bars are ± standard deviation.

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Desensitization of the de novo mediators secretion by exposure to sub-threshold FceRI clustering In view of the above results, we were interested in examining the possibility of desensitizing the secretory response as expressed in that of de novo synthesized inflammatory mediators (e.g. TNFa and IL4) under conditions similar to those employed for desensitizing the degranulation. In order to discriminate between the cells’ basal TNF-a content, shown by previous studies (Young et al 1987) to be present even in untreated RBL-2H3 cells, and the de novo synthesized and secreted product upon FceRI clustering, we employed both desensitization and stimulation periods varying between 4 and 14 hours. The threshold and optimal concentrations of antigen were determined as previously described at each of the employed concentrations of monomeric IgE (0.05, 0.5, 5 or 50 nM). Cells were then usually stimulated for 14 hours with a range of antigen concentration (0.1–100 ng/ml). Significantly, the antigen concentrations sufficient for inducing cytokine secretion were found to be markedly lower than those required for inducing degranulation and respectively, the sub-threshold antigen concentrations were found to be three- to sixfold lower than those used for inducing the desensitization of the degranulation. Both desensitization and optimal antigen concentrations were also found to depend on the employed IgE concentrations (e.g. optimal secretion was caused by 30 ng/ml at 50 nM IgE or 10 ng/ml at the other IgE concentrations) (data not shown). The relation between the desensitizing antigen concentrations and the length of the desensitization period reflected in the decrease of secreted TNFa and IL4 was also investigated. To this end, IgE primed cells were desensitized by incubation with a range of sub-threshold antigen concentrations (0.045 to 1.5 ng/ml) for periods between 4 and 14 hours. We then determined and subtracted the amounts of new products synthesized and secreted during this desensitization period and proceeded with post-desensitization stimulation using optimal concentrations of antigen. The secreted TNFa and IL4 were assayed again. Results showed that the desensitization caused a marked decrease in secretion of de novo synthesized cytokines. This depended on all three examined parameters: 䊉

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The concentration of the monomeric IgE: the largest decrease (65–85%) in the secretion was observed when lowest IgE concentration (0.05 nM) was employed while at 5 nM IgE the magnitude of desensitization ranged between 13% and 55% (Fig. 4A and 4B); The desensitizing Ag concentrations: The highest desensitizing concentrations within the range were always found to be the most effective, independent of the IgE concentration (Figs 4 and 5); The length of the incubation period: the longer the desensitization period the larger the decrease in the response (Fig. 5A and 5B).

FIG. 4. Sub-threshold FceRI clustering induces desensitization of TNFa secretion. Adherent RBL-2H3 cells (1 ¥ 105/100 ml/well) were reacted with 0.05 nM (A), or 5 nM (B) of monomeric A2IgE for 2 hours. Following washings in DMEM, the cells were incubated for 14 hours with the indicated desensitizing antigen concentrations. Control cells were kept in DMEM for the same period of time. Desensitized and non-desensitized cells were next stimulated for another 14 hours with the optimal antigen concentrations, adjusted to the employed concentration of the monomeric IgE: 30 ng/ml (A) or 100 ng/ml (B). Amount of secreted TNFa was determined by ELISA, using an assay kit (R&D Systems, USA) according to the manufacturer’s instructions and presented as function of the antigen desensitizing concentrations. The desensitizationdependent decrease of the secretory response is shown on the right ordinate. Data are from one experiment out of two and points represent the average of triplicates. Error bars are ± standard deviation.

FIG. 5. Sub-threshold FceRI clustering induces desensitization of IL4 secretion. RBL-2H3 cells (1 ¥ 105/100 ml/well) were first incubated for 2 hours with 0.05 nM of the A2IgE monomeric. Cells were then washed with DMEM and desensitized for 14 (A) or 8 hours (B) with the indicated antigen concentrations. Control cells were maintained in DMEM for the same periods of time. Desensitized and non-desensitized cells were washed and stimulated with the optimal concentration of antigen (10 ng/ml) for the same periods of time as those used for the desensitization. Cells’ secretory response was assayed by ELISA using an IL4 kit from R&D Systems, USA according to the manufacturer’s instructions. The secretion is presented as function of the desensitizing antigen concentration. The desensitization-dependent decrease in the IL4 secretion is indicated on the right ordinate. Data are from one representative experiment out of three and shown values represent average of quadruplicates. Error bars are ± standard deviation.

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In conclusion, results of these experiments clearly show that exposing mast cells to sub-threshold concentrations of FceRI clustering agents for prolonged time periods induces marked reduction of their secretory response upon subsequent exposure to optimal stimulation. As these experiments were performed on the welldefined RBL-2H3 cell line and employing quantitatively characterized reagents, the results provide a first step towards a rigorous analysis of the process. Clearly, understanding the biochemical mechanism underlying this desensitization is now the challenge. Preliminary results indicate that the desensitization protocols induce a considerable degree of endocytosis of the cell-resident IgE. The questions as to whether this is indeed the main cause for the decrease in the cellular response and whether other receptor-proximal events in the FceRI stimulus-response coupling cascade are involved in this form of desensitization are currently being investigated. References Abramson J, Rozenblum G, Pecht I 2003 Dok protein family members are involved in signaling mediated by the type 1 Fc epsilon receptor. Eur J Immunol 33:85–91 Barbu EA, Licht A, Pecht I 2002 Control of mast cells’ secretory response to the Fc epsilon receptor stimulus: is there desensitization? Isr Med Assoc J 4:874–875 Blaser C, Kaufmann M, Pircher H 1998 Virus-activated CD8 T cells and lymphokine-activated NK cells express the mast cell function-associated antigen, an inhibitory C-type lectin. J Immunol 161:6451–6454 Butcher S, Arney KL, Cook GP 1998 MAFA-L, an ITIM-containing receptor encoded by the human NK cell gene complex and expressed by basophils and NK cells. Eur J Immunol 28:3755–3762 Cambier JC 1995 New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL). Immunol Today 16:110 Daeron M, Malbec O, Latour S, Arock M, Fridman WH 1995 Regulation of high-affinity IgE receptor-mediated mast-cell activation by murine low-affinity IgG receptors. J Clin Invest 95:577–585 Guthmann MD, Tal M, Pecht I 1995 A secretion inhibitory signal transduction molecule on mast cells is another C-type lectin. Proc Natl Acad Sci USA 92:9397–9401 Ishizaka T, Sterk AR, Daeron M, Becker EL, Ishizaka K 1985 Biochemical analysis of desensitization of mouse mast cells. J Immunol 135:492–501 MacGlashan D Jr, Lichtenstein LM 1987 Basic characteristics of human lung mast cell desensitization. J Immunol 139:501–505 Nakamura M, MacGlashan D Jr 1994 Desensitization of IL-4 secretion from mouse bone marrow-derived mast cells. Immunol Lett 41:129–133 Ortega SE, Pecht I 1988 A monoclonal antibody that inhibits secretion from rat basophilic leukemia cells and binds to a novel membrane component. J Immunol 141:4324–4332 Reth M 1989 Antigen receptor tail clue. Nature 338:383–384 Sobotka AK, Dembo M, Goldstein B, Lichtenstein LM 1979 Antigen-specific desensitization of human basophils. J Immunol 122:511–517 Weetall M, Holowka D, Baird B 1993 Heterologous desensitization of the high affinity receptor for IgE (Fc epsilon R1) on RBL cells. J Immunol 150:4072–4083 Xu R, Abramson J, Fridkin M, Pecht I 2001 SH2 domain-containing inositol polyphosphate 5¢phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen. J Immunol 167:6394 –6402

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Xu R, Pecht I 2001 The protein tyrosine kinase syk activity is reduced by clustering the mast cell function-associated antigen. Eur J Immunol 31:1571–1581 Young JD, Liu CC, Butler G, Cohn ZA, Galli SJ 1987 Identification, purification, and characterization of a mast cell-associated cytolytic factor related to tumor necrosis factor. Proc Natl Acad Sci USA 84:9175–9179

DISCUSSION MacGlashan: A few years ago we switched from looking at desensitization in the context of the absence of Ca2+ to doing a full secretion model. The dynamics of desensitization are changed when there is a Ca2+ response as part of the reaction. A recent paper looks at this suboptimal desensitization in the human basophil (MacGlashan & Miura 2004). What appears to be happening is that we get an integrative response, where other parameters at low concentration of antigen don’t seem to elicit much of a change. We get measurable changes in the phosphorylation of c-Cbl and a progressive but slow down-regulation of Syk kinase. We provide some correlative data that suggest there is ubiquitination of Syk. If that is the case, it seems likely that there are other components as well that are lost, on the basis of work done in rat basophilic leukaemia (RBL) cells. This may explain the severity of the loss of functional response. Galli: I would like to return to the earlier discussion about the potential relationship of aggregation of the FceRI receptor with the low-level release of mediators. In this situation, the cell exhibits a diminished functional response after prolonged incubation with IgE and low concentrations of antigen. What are your thoughts about the apparently disparate findings of low level stimulation through the receptor either producing cytokines or reducing their production? Also, a technical question: during the 12–14 h incubation with low levels of antigen, was there any evidence of signalling? Austen: Israel Pecht, before you answer these questions, let’s clarify something: did you have Ca2+ in there or not? Pecht: Of course we have Ca2+. We did all these experiments under secretionpermissive conditions, i.e. with the right concentration of Ca2+. In response to Dr Galli’s questions, we looked first at what mediators and how much of each is being secreted, primarily in terms of cytokines, during the relatively long desensitization periods but have also examined the situation for the degranulation. Thus, there are, of course, signalling events being triggered and these have also been investigated. Depending on the particular desensitization agent concentration we use and the mediator monitored, we get a commensurate response to the so-called ‘subthreshold’ stimulation. This is less of a problem with the degranulation because of the immediate response. In the case of cytokine secretion, we have quantitatively determined it following both the desensitization and of course after stimulation and the appropriate corrections were introduced.

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With regard to the first point that you made, we spent quite some time looking at what monomeric IgE is doing under these conditions. Our whole protocol was based on using strictly purified monomeric IgE. Whenever we have used ‘better’ stimulating monomeric monoclonal IgEs such as the SPE-7, we encountered the published problem as well. Hence, we have avoided the use of this one. In other words, we can sort out the two problems and deal with them independently. Kawakami: Following Steve Galli’s comment, we should be aware that after SPE-7 incubation, of course we can add antigen and measure degranulation, but the extent of degranulation and also cytokine production is rather lower after antigen stimulation if we compare this to activation just by SPE-7. This is similar to the desensitization after stimulation with IgE and antigen. Brown: Do you have evidence that you are actually getting inhibition of new cytokine synthesis with mRNA transcription? Pecht: Yes, both were measured, although not for the whole list I showed. This is true for IL4 and TNFa. Brown: Both of these are known to be expressed constitutively by some of the RBL cells we have worked with. Pecht: As stated in my response to earlier questions, we have always determined secretion values induced during the relatively long desensitization process as well as those induced by the actual stimulation and the basal levels. Hence we are confident about the values determined following the induced desensitization. Stevens: The expression of many cytokines is regulated at the mRNA level by transcriptional and post-transcriptional mechanisms. Thus, have you carried out a nuclear run-on assay to see if the difference in cytokine mRNA levels is due to variable transcription or variable catabolism of the initially expressed transcript? Pecht: Not yet. Stevens: Many cytokine transcripts that are regulated by a post-transcriptional mechanism possess an ‘AUUUA’ motif in their 3¢ untranslated regions (Shaw & Kamen 1986). Do any of the cytokines that you are investigating possess this motif ? Pecht: We haven’t checked this. Ono: Going back to the first part of your paper, can you say more about MAFA and its physiological ligand? Pecht: The ligand is still evasive. We’ve made considerable efforts to look for it. As expected, we found that the C-lectin domain of MAFA binds terminal mannose residues. This is therefore one bona fide ligand. I don’t believe, though, that this is the physiological ligand. One reason is that we have so many analogues of MAFA, such as CD23, which contain C-type lectin domains, and do not interact with their known ligands via carbohydrate epitopes. This is as far as I can illuminate the issue of the ligand. There are homologues of MAFA in mouse and human, but they are not typical mast cell membrane components. In the mouse MAFA isn’t found

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on mast cells but is rather expressed by NK cells and specific sets of T cells. This is why we believe MAFA has a broader immunological significance than we have so far described in the mast cells. In the human it is expressed on mast cells also. Very little is known about it so far. Some of our results suggest that a ligand may be involved in adhesion as clear correlation between MAFA levels expression and the cells’ adhesiveness was observed ( J. Abramson and I. Pecht, unpublished results). Rivera: One of the interesting features is that you are getting selective cytokine inhibition. Looking at the list, it did not look like it was Th1 or Th2 specific. What do you think the function of such a molecule might be if you are not downregulating or dampening a Th1 or Th2 response? Pecht: I don’t know. It might be an aberration. Still, for instance we get no inhibition of IL5 and IL3. These are known to induce growth and maturation of eosinophils and it may therefore seem that MAFA does not modulate the inflammatory response at this particular level. Similarly, another unaffected molecule is IL16 which is known as a chemokine with selective chemoattraction for CD4+ inflammatory T cells, suggesting that MAFA will not affect recruitment of these cells to the site of inflammation. In contrast, molecules like IL4 and IL8 are significantly affected. This may potentially suppress the recruitment of eosinophils and neutrophils or differentiation of T cells into the Th2 subset. Rivera: You think Dok1 and SHIP1 are involved in this. What happens in terms of PIP3 production? Pecht: There is a clear decline in PIP3 levels and we have found this quite some time ago, during the early phase of characterizing MAFA’s mode of action. Dok-1 is clearly involved in suppression of the Erk signalling pathway. Of course, selective inhibition of MAP kinases would likely result in a selective inhibition of the synthesis of cytokines. Koyasu: In both humans and mice, NK cells express MAFA. Can you inhibit NK cell activity by cross-linking MAFA? Pecht: That is not our work, but of Pircher and colleagues (Voehringer et al 2002). I don’t remember how they did it. In our case, it’s important to note that there is no need for co-clustering MAFA with the FceRI to get an inhibitory effect. Significantly, in recent studies we have shown that a marked increase in inhibition is observed upon co-clustering (Licht et al 2005). Stevens: I am surprised that rats and humans express MAFA but not mice. Mouse mast cells are heterogeneous in tissues. Thus, did you examine different populations of mouse mast cells for their expression of MAFA? Is there a mutation or deletion in the mouse’s genome that prevents MAFA expression? Lastly, mouse strains have been identified that differ in their mast cell protease expression. Thus, have you examined MAFA expression in different mouse strains?

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Pecht: Again, this isn’t our work. I can’t answer you. Kawakami: We have evidence that mouse bone marrow derived mast cells (BMMCs) don’t express it. Stevens: IL3-developed mBMMCs phenotypically are very different from cutaneous mast cells. For example, calcium ionophore-activated mBMMCs preferentially metabolize arachidonic acid to leukotriene C4 whereas activated cutaneous mast cells preferentially produce prostaglandin D2. I don’t know another example where a gene is expressed in rat and human mast cells but not mouse mast cells. Rao: Going back to the desensitization question, you have these two forms: do you have any idea of the relationship between them? Pecht: We had only one form. Rao: What about the desensitization that happened without Ca2+? Pecht: That is not our work. We intentionally avoided using the protocol under these secretion non-permissive conditions such as those avoiding Ca2+ in the medium. We wanted to employ conditions which are as close to the physiological ones as we could get. We have used both antigen and an IgE-specific mAb as clustering agents, and in both cases the same pattern of desensitization has been observed. The idea is to identify the highest concentration of FceR clustering agent that is not causing secretion and use this for a prolonged, usually overnight treatment and characterize the observed desensitization pattern. Rao: Is this desensitization dependent on outside Ca2+? Pecht: Yes. Rao: Are you aware of the phenomenon that has for a long time been known as T cell anergy? This is a Ca2+-dependent process down-regulating the T cell receptor (TCR) responses. Pecht: Yes. This is why one of the first parameters we checked was whether the surface expression levels of the FceR and IgE are affected. We have found that there is no quantitative or even qualitative correlation between the limited decline in the binding of IgE or in the expression levels of FceRI on the cells’ surface and the observed desensitization. As Don MacGlashan mentioned earlier, Syk is one component that is decreasing in concentration during this process. Rao: In T cells, anergy is actually the consequence of a complicated transcriptional feedback loop that involves activation of a transcription factor called NFAT. This then turns on genes whose products down-regulate signalling in the T cell. Is something like this operating in the mast cells? It is easy to check. For instance you could check mast cells that don’t have this transcription factor, and if the hypothesis is correct they should not undergo this desensitization. MacGlashan: It happens rapidly. The event we are seeing is tuned way back. Everything you see happening at that low level you can see happening at the high level; it just happens much faster. There are several reasons we got into this. From the

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perspective of clinical therapy, there is clinical desensitization. The idea is to ramp up the exposure of the patient to antigen that they are sensitive to, starting at very low concentrations. The idea in our experiments is to stimulate cells at a low level. Can we induce this state of ‘anergy’ in the cell in the absence of it having ever secreted? This is what Israel Pecht is trying to set up, and what we have set up as well. In fact, we can do this. This system is sensitive in a way that the cell can turn itself off without much secretion. Steve Galli asked about cytokine secretion. We haven’t touched on this, but in terms of fast mediators such as histamine and leukotrienes there isn’t any secretion. Since we have been studying this as a continuum process, I can’t say for sure whether or not it is something different from what we see happen clinically, but I suspect it is a very similar process. With respect to the Ca2+ issue, other than the fact that a cell that is under full secretion has a long-lived Ca2+ response, we have never been able to pick up a distinction there. The same basic processes seem to occur one way or another. We switched to not using the operational method of eliminating Ca2+ solely because we wanted to maintain the dynamics seen under full secretion. As far as I can tell from studies of the two types there is no marked signalling difference. Pecht: Coming back to Anjana Rao’s point about the T cells, how is this state of anergy being induced? Is it being induced also by the absence of Ca2+? Rao: No. Pecht: Then it is a different story altogether. Rivera: One of the things that is intriguing is that if you are suggesting that Syk is a key regulator or desensitizer in the system, presumably there still has to be receptor phosphorylation at some point in order for Syk to become active and become ubiquitinated. Is this like Paul Allen’s or Ron Germain’s studies with anergic peptides, where there is differential phosphorylation of receptor giving different signals towards activating Syk? MacGlashan: It is certainly possible. The problem is that as you drop off the strength of the stimulus you no longer see things happening that you normally would. Under those circumstances we might not be looking at the pathway that is particularly active, so it looks like everything is at a very low response level. However, when we looked at Cbl phosphorylation, this has a sensitivity similar to the desensitization sensitivity. In other words, that molecule can be phosphorylated at levels of stimulation for which you don’t see Syk phosphorylation or any of the downstream phosphorylation. Since we can see the association between Syk and Cbl, it acts as if the system is integrating a very low level of stimulus of Syk, Cbl gets phosphorylated and never gets dephosphorylated. It therefore integrates the response, and begins ubiquitinating the Syk and the whole thing shuts down. Pecht: This model is quite different from the one that is currently accepted for the T cell anergy situation, where there is for example a rather different pattern of zeta chain phosphorylation.

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Razin: What do you mean by saying that MAFA is not expressed on mouse mast cells? Is this the outer membrane you are referring to? Pecht: It isn’t present at the mRNA level, either. Razin: Did you check in the rat mucosa mast cells? Pecht: Yes, it is expressed there. References Licht A, Pecht I, Schweitzer-Stenner R 2005 Regulation of mast cells’ secretory response by coclustering the Type I Fepsilon receptor with the mast cell function-associated antigen. Eur J Immunol 35:1621–1633 MacGlashan D, Miura K 2004 Loss of syk kinase during IgE-mediated stimulation of human basophils. J Allergy Clin Immunol 114:1317–1324 Shaw G, Kamen R 1986 A conserved AU sequence from the 3¢ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659–667 Voehringer D, Koschella M, Pircher H 2002 Lack of proliferative capacity of human effector and memory T cells expressing killer cell lectin-like receptor G1 (KLRG1). Blood 100:3698–3702

General discussion I

Ono: I’d like to begin this general discussion by asking a question about MITF. I understand that MITF is extremely important for transcription of the PGD2 synthase gene. How is the RasGRP4 signalling moving through MITF? Is it inducing MITF or is it modifying it such that MITF-dependent genes are activated? Stevens: It is now apparent that MITF is regulated in a complex manner. At least five MITF isoforms have been identified in the body that are the result of differential splicing of the MITF precursor transcript (Takemoto et al 2002). Complicating the situation is the fact that MITF’s activity can be altered if this transcription factor is post-translationally modified by SUMO (Miller et al 2005) or phosphate (Sonnenblick et al 2004). RasGRP4 and its downstream factors probably induce the phosphorylation of cytosolic MITF which enables the transcription factor to dissociate from its inhibitor(s). Razin: I agree that it is a complex system. The regulation of MITF could be at the level of phosphorylation, or the dissociation of MITF from its inhibitors (we now know of two inhibitors, one of which is Hint and the other is an inhibitor of STAT3), or it could be through its association with its activators. Even the phosphorylation is not simple, because phosphorylation at serine 409 of MITF causes dissociation of one of the inhibitors of MITF. Kitamura: Also, MITF is involved in the transcription of Kit. It is located not only downstream but also upstream of Kit. Ono: Rick Stevens, have you checked whether it is phosphorylated at rest or whether there is an up-regulation after transduction of RasGRP4? Stevens: It remains to be determined if RasGRP4 is phosphorylated inside c-kitactivated mast cells, but RasGRP3 is phosphorylated in HEK-293 cells when these transfectants are activated via their epidermal growth factor receptors (Stope et al 2004). Whether or not RasGRP4 itself is phosphorylated, it is likely that this guanine nucleotide exchange factor controls downstream phosphorylation events in c-kit-activated mast cells because MITF is phosphorylated in c-kit activated mast cells. It is believed that the phosphorylation of MITF is needed for its dissociation with HINT and its translocation from the cytoplasm into the nucleus. Austen: I enjoyed Juan Rivera’s paper. I think the finding of negative regulation is compatible with the fact that inflammation needs constitutive regulation. Bad events occur not just with activation but also when the controls are gone. Juan, can you say something more about PKCq? There is a lot of literature on this focused 95

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on the T cell, including Tregs, T memory cells and natural killer T lymphocytes (NKTs). The other cell of the haematopoietic lineage is the mast cell. Rivera: Our knowledge on PKCq in the mast cell system is quite limited. The studies we have done have analysed the overexpression of PKCq in rat basophilic leukaemia (RBL) 2H3 cells as well as a dominant-negative mutant of PKCq. We have looked at functional aspects and have some results on localization as well. Unlike in the T cell receptor (TCR), where it looks like PKCq is part of the synapse, it does not appear that PKCq is localized to the aggregates of IgE receptor, although we haven’t done those experiments in the real sense of forming a large cluster to see whether PKCq is there. We can detect PKCd in the receptor complex in that context. Part of our thinking is that perhaps PKCd is doing the same thing as PKCq in the IgE receptor context. In unpublished studies we have analysed the PKCq knockout mice, and couldn’t find a dramatic alteration of phenotype, except for the same things that we saw in our overexpression experiments. This was a pretty selective role on interleukin (IL)6 production. It is not a dramatic phenotype in that there are still significant amounts of IL6 being produced. We didn’t exhaustively look at all mast cell functional responses. It could be that we didn’t analyse the appropriate leukotrienes or cytokines, so we can’t conclude that it doesn’t play a significant role. It is clearly present and gets activated in response to IgE receptor. Austen: Did you say PKCd is there? This is relevant because PKCd has the opposite effect, and knocking it out causes B cells that don’t die. Rivera: The thing we know about PKCd is that it interacts with Lyn. This interaction seems to increase the activity of Lyn. In the absence of this interaction we see decreased Lyn activity, so it seems that d is regulating Lyn activity and upregulating the negative role of Lyn in the cells. Now that we know some of the outcomes of Lyn-negative regulation, it may be important to go back and analyse whether those outcomes are exacerbated by PKCd in some way. Kawakami: We had a chance to analyse the same mice. We didn’t see any significant effects of degranulation or cytokine production (we looked at tumour necrosis factor [TNF]a, IL6 and IL2). Also, we looked at similar signalling events, which aren’t dramatically affected. This is why we didn’t publish this. Galli: Is it clear in both of these studies that no compensation occurred as a result of the absence of this PKCq isoform? Kawakami: The expression level of PKCq isn’t very high in bone marrow-derived mast cells (BMMCs). We measured the expression level of several PKC isoforms such as a (40 ng/one million cells), b1 (1.25 ng/one million cells) and b2 (50 ng/one million cells). The level of q is 8 ng/one million cells. Rivera: We didn’t see any compensatory effect of down-regulation of theta. Metcalfe: I wanted to comment on something Rick Stevens said about HMC1 cells being hypogranular. It is possible that activating mutations in Kit might lead to some constitutive degranulation. At least in humans, when we isolate mast cells with the


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Kit codon 816 mutation, they look fairly normal. This doesn’t mean that they are not releasing mediators, but they are not obviously hypogranular. They do often express some other features that are aberrant, such as the a chain of the IL2 receptor, CD25. You mentioned that stem cell factor (SCF) is a degranulating agent. It is, but this brings up a conundrum. When we do in vitro modelling with primary mast cell cultures, we may study these cells either in the presence or absence of various growth factors. For instance if mouse BMMCs are cultured with IL3 and then SCF is added, you can get dramatic effects, some of which go away when SCF is removed. In humans this approach cannot be used because it isn’t possible to grow human mast cells in IL3 and then bring in SCF. You have to grow human mast cells in SCF, then deprive them of SCF. And when it is added back you can show that this addition of SCF will change cytokine profiles due to IgE-mediated mast cell activation (Hundley et al 2004). Thus there is the whole issue of whether mast cell biology can be interpreted out of the context of normal stimulation by growth factors. In humans the normal level of soluble SCF is about 800 pg/ml (Brockow et al 2002), but the concentrations reached when a mast cell is nestled against a stromal cell can be quite different, as can the effects. Austen: Is that protein, or function? Metcalfe: It’s protein. Austen: It would be important for someone to do a bioassay before we conclude that these levels are real. Metcalfe: I think they probably are real, but these levels may not reflect the tissue situation in any case. Austen: I’d still like to see you do a dose–response study. Metcalfe: We’ve attempted this, but we were limited by the problem of deprivation. If we look in mouse mast cells with SCF we can show adhesion with addition of very low levels of SCF, suggesting these serum levels of SCF are sufficient to induce mast cell adhesion to matrix, thus the lack in normal biology of circulating mast cells (Dastych & Metcalfe 1994). Austen: I am talking about the human levels of SCF in the circulation. Metcalfe: As human mast cells are cultured in SCF, these mouse experiments are difficult to reproduce using human mast cells. What ultimately counts is the presentation of SCF in the microenvironment. Koffer: I was thinking about the Lyn-deficient cells. You said that Syk is partially activated in them: how? Second point: is this partial activation sufficient to activate the normal pathway downstream of Lyn? Rivera: We don’t know the mechanism for the partial activation. However, we do get some receptor phosphorylation even in the absence of Lyn. Presumably, what one sees as activation of Syk, which is dramatically delayed in these cells and is lower overall, happens after some time because the receptor is still phosphorylated. Downstream of this, phosphorylation of LAT, Vav1 and SLP-76 is all defective,

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but these are partial defects. It is clear that this partial signal is still required for degranulation. We have pretty good data on this. In Lyn deficiency, Ca2+ responses are delayed, and we never see a recovery of this response to the extent of normal cells, although it is a sustained response. If we take those cells and incubate them with an intracellular Ca2+ chelator, degranulation is ablated. Some signal is therefore needed through this pathway for degranulation to occur. Our perspective is that while both arms are needed, the Fyn arm is driving the degranulation response. When we look at the total phosphatidylinositol-3-kinase (PI3K) activity found in the wild-type versus the Fyn-deficient cells, it is reduced by about 80%. If one uses the PI3K inhibitor and looks at the degranulation of wild-type cells and Lyn-deficient cells, it is very effective at inhibiting the degranulation response. Thus, the PI3K arm seems to be central, although some Ca2+ signals are needed still. Kawakami: Juan Rivera, you showed a nice story about a negative regulatory pathway for Lyn. We also have to be aware that Lyn plays a positive role—it depends on the outcome that you are looking at. Lyn plays a positive role in cell adhesion to fibronectin. The same is also true for IgE- or IgE plus antigen-induced migration. Therefore, there are both positive and negative aspects of Lyn function. Rivera: I agree. The read-out is important in terms of whether Lyn plays a positive versus a negative role. Our data on adhesion are similar to Toshi’s. With chemotaxis our data are different, but this may be because of the different systems we are using. Toshi is using fibronectin coating, but we decided not to use this, because we were worried about activating integrin signalling. This could have compensatory mechanisms for Lyn function. When we do chemotaxis without fibronectin, Lyn deficient cells show normal chemotaxis. I am not sure how to interpret this result. In terms of the role for Lyn, everything we have measured, i.e. chemokines, cytokines and degranulation, is up-regulated. Importantly, however, in our most recent studies where we looked at the association of Lyn with the IgE receptor, this phenotype looks different from the total Lyn knockout. It is important to keep in mind that the regulatory control that Lyn is exerting on degranulation does not appear to be mediated by receptor-associated Lyn and receptor-associated Lyn is playing a positive role in degranulation. Mechanistically, we don’t understand this yet. Razin: Is it possible that you are a talking about a few isoforms of Lyn? Rivera: In the knockout both the A and B form are missing. They are alternately spliced isoforms and both are gone. In the human system we have targeted the B form. However, both isoforms are gone in the human system, also. Thus, our data do not distinguish isoforms. Koyasu: When you say chemotaxis, which chemokine did you use? Rivera: We have done several readouts with a range of ligands. We have used Ig antigen as a ligand, and also S1T and SEF.


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Ono: Israel Pecht, have you looked at the impact of MAFA aggregation on chemotaxis? Pecht: No, but this definitely deserves to be tested. References Brockow K, Akin C, Huber M, Scott LM, Schwartz LB, Metcalfe DD 2002 Levels of mast cell growth factors in plasma and in suction skin blister fluid in adults with mastocytosis: correlation with dermal mast cell numbers and mast cell tryptase. J Allergy Clin Immunol 109:82–88 Dastych J, Metcalfe DD 1994 Stem cell factor induces mast cell adhesion to fibronectin. J Immunol 152:213–219 Hundley TR, Gilfillan AM, Tkaczyk C, Andrade MV, Metcalfe DD, Beaven MM 2004 Kit and Fce RI mediate unique and convergent signals for release of inflammatory mediators from human mast cells. Blood 104:2410–2417 Miller AJ, Levy C, Davis IJ, Razin E, Fisher DE 2005 Sumoylation of MITF and its related family members TFE3 and TFEB. J Biol Chem 280:146–155 Sonnenblick A, Levy C, Razin E 2004 Interplay between MITF, PIAS3, and STAT3 in mast cells and melanocytes. Mol Cell Biol 24:10584 –10592 Stope MB, Vom Dorp F, Szatkowski D et al 2004 Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3. Mol Cell Biol 24:4664 –4676 Takemoto CM, Yoon YJ, Fisher DE 2002 The identification and functional characterization of a novel mast cell isoform of the microphthalmia-associated transcription factor. J Biol Chem 277:30244 –30252

IgE regulation of mast cell survival and function Toshiaki Kawakami, Jiro Kitaura, Wenbin Xiao and Yuko Kawakami Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, California 92121, USA

Abstract. Traditionally, it is thought that IgE binding to mast cells via the high-affinity receptor (FceRI) is simply a passive ‘sensitization’ step prior to activation by receptor aggregation or cross-linking with multivalent antigen or other cross-linking agents. However, in addition to receptor up-regulation, recent studies have shown that monomeric IgE can induce survival and other activation events including increased histamine content, degranulation, leukotriene release, receptor internalization, adhesion, migration and DNA synthesis. Various IgE molecules exhibit a vast spectrum of heterogeneity: the highly cytokinergic (HC) group of IgEs at an extreme end of the spectrum can induce survival and other activation events very efficiently, whereas poorly cytokinergic (PC) IgEs at the other end can do so less efficiently. All the IgEs tested appear to be capable of inducing receptor aggregation with HC IgEs having a higher capacity to do so than PC IgEs. HC IgEs can promote the production and secretion of various cytokines including the one(s) that can sustain survival in an autocrine and paracrine mechanism. Consistent with receptor aggregation induced by monomeric IgE, other means of receptor aggregation, e.g. IgE+antigen and IgE+anti-IgE, can also induce survival and other events in unique ranges of stimulation intensity. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 100–114

Mast cells are major effector and regulatory cells for immediate hypersensitivity and allergic diseases. Cross-linking of IgE bound to its high-affinity receptors, FceRI, with multivalent antigen (the stimulation mode hereafter termed IgE+Ag) initiates the activation of mast cells by promoting the aggregation of FceRI (Metzger 1992, Turner & Kinet 1999). This FceRI-dependent activation results in degranulation (secretion of preformed mediators that are stored in the cytoplasmic granules, such as vasoactive amines, neutral proteases and proteoglycans), the de novo synthesis of proinflammatory lipid mediators, and the synthesis and secretion of cytokines and chemokines (Galli 1999). These chemical and peptide mediators contribute to the development of allergy and other forms of inflammation. The FceRI on murine mast cells consists of four subunits: an IgE-binding a subunit, a signal-amplifying, receptor-stabilizing b subunit and two disulfide-bonded 100

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g subunits that are the main signal transducer (Kinet 1999). Aggregation of FceRI induced by IgE+Ag or IgE+anti-IgE (stimulation of IgE-sensitized cells with antiIgE antibody) leads to the activation of this system: b subunit-associated Lyn, a Src family protein-tyrosine kinase (PTK), becomes activated and phosphorylates tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic regions of b and g subunits (Turner & Kinet 1999). Phosphorylated b and g ITAMs recruit Lyn and Syk (another PTK with two tandem Src homology [SH]2 domains N-terminal to the catalytic domain), respectively. Another Src family PTK, Fyn, was recently shown to associate with FceRI and to play a complementary role, particularly by activating phosphatidylinositol-3-kinase (PI3K) (Parravicini et al 2002). These PTKs phosphorylate numerous targets and activate several signalling pathways, including the PI3K, phospholipase C/Ca2+ and several mitogen-activated protein kinase (MAPK) pathways (Turner & Kinet 1999, Kawakami & Galli 2002). These signalling events lead to degranulation and cytokine production. In this contribution, we will review recent progresses on biological effects of monomeric IgE on mast cells and their mechanisms.

FceRI up-regulation by IgE A few papers published in 1977–1978, which have proved very important in retrospect, demonstrated that there is a positive correlation between serum IgE levels and IgE-binding capacity of basophils (hence FceRI expression levels) in atopic patients (Stallman et al 1977, Conroy et al 1977, Malveaux et al 1978). This phenomenon was shown in cultured mast cells in 1985 (Furuichi et al 1985, Quarto et al 1985) and revisited in 1996–1997 (Hsu & MacGlashan 1996, Yamaguchi et al 1997). Progresses made in the latest wave of studies demonstrated that the enhanced surface expression of FceRI by IgE renders mast cells and basophils sensitive to lower antigen concentrations and levels of mediators released upon activation are increased in cells with higher levels of FceRI. Receptor up-regulation was shown to be due to the stabilization and accumulation of FceRI on the mast cell surface in the presence of continued basal levels of protein synthesis (Borkowski et al 2001, Kubo et al 2001).

‘Monomeric’ IgE promotes mast cell survival The two studies showed that mouse mast cell survival and growth are promoted by monomeric IgE binding to its receptor, FceRI (Asai et al 2001, Kalesnikoff et al 2001). Monomeric IgE suppresses the apoptosis induced by growth factor deprivation. This anti-apoptotic effect does not depend on IgE-induced FceRI

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up-regulation, but requires the continuous presence of IgE. These studies, however, suggest differences in the potential mechanisms: Kalesnikoff et al (2001) found that IgE binding induces secretion of a variety of cytokines that enhance cell survival by an autocrine mechanism. In support of their model, they also found tyrosine phosphorylation of FceRI b subunit and activation of Akt and MAPKs in IgE-treated mast cells. By contrast, Asai et al (2001) did not detect significant cytokine secretion or any signaling events, which are known to be elicited by FceRI aggregation, in IgE-treated mast cells. Despite the differences, these observations transformed the traditional view of IgE–mast cell binding as a passive ‘sensitization’ step prior to receptor aggregation with antigen or other cross-linking reagents into a new one that monomeric IgE can actively induce survival and activation of mast cells (Kawakami & Galli 2002). Kitaura et al (2003) subsequently demonstrated that all of the IgE molecules tested showed anti-apoptotic effects on mast cells, but that the different IgEs exhibited a wide spectrum in their ability to induce the production and secretion of cytokines by mast cells. At one extreme, highly cytokinergic (HC) IgEs can induce strong anti-apoptotic effects, in part by an autocrine mechanism. At the other end of the spectrum, poorly cytokinergic (PC) IgEs induce less robust survival effects, but without inducing detectable cytokine production. Importantly, several lines of evidence, particularly the one generated by time-resolved phosphorescence anisotropy measurements, indicate that binding of either HC or PC IgEs can result in FceRI aggregation in the absence of antigen for which that IgE is known to have specificity, with more extensive FceRI aggregation induced by HC than by PC IgEs. Therefore, Kitaura et al (2003) could explain the apparent disparity in the above two studies. Furthermore, mast cell numbers were increased in some gastrointestinal mucosal tissues of mice harbouring IgE-secreting hybridoma, consistent with the survival effect of high serum IgE levels. Some concerns have been raised during the course of the above studies: (1) are the survival and other effects due to ‘monomeric’ IgE, but not aggregates contaminated in IgE preparations? (2) Are the effects due to other contaminant(s) such as endotoxin? (3) How can monomeric IgE induce receptor aggregation? Usually, IgE preparations contain small quantities of aggregates and can be mostly removed by ultracentrifugation or gel filtration. Monomeric IgE preparations highly purified by these procedures exhibited survival and other effects, and aggregates present before extensive purification have less or similar capacity to exert such effects than the purified monomeric IgEs (Kalesnikoff et al 2001, Oka et al 2004). Other contaminants such as endotoxin cannot be a major effector molecule because IgE preparations could not promote survival in mast cells lacking FceRI (Asai et al 2001). Therefore, it is without doubt that monomeric IgE can induce survival and other activation effects.

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‘Monomeric’ IgE induces various activation events similar to two other stimulation modes, i.e. IgE+Ag and IgE+anti-IgE Following the two studies by Asai et al (2001) and Kalesnikoff et al (2001), numerous groups of researchers have studied effects of monomeric IgE on mast cell biology. Other activation events that have been shown to be induced by monomeric IgE include increased histamine content (Tanaka et al 2002, Kitaura et al 2003), cell adhesion to fibronectin (Lam et al 2003), migration (Kitaura et al 2005), Ca2+ flux (Pandey et al 2004), membrane ruffling (Pandey et al 2004), histamine release (Kitaura et al 2003, Pandey et al 2004), leukotriene release (Kitaura et al 2003), receptor internalization (Kitaura et al 2003), DNA synthesis (Kitaura et al 2003), and sensitization to compound 48/80 and substance P (Yamada et al 2003). These findings are not surprising in light of our observation that all IgE molecules can induce receptor aggregation (Kitaura et al 2003). This also suggests that signalling events transduced by different modes of receptor aggregation, i.e. monomeric IgE, IgE+Ag and IgE+anti-IgE, may be similar, if not identical. However, the intensity of stimulation induced by these different means of receptor aggregation seems different among them. For example, tyrosine phosphorylation can hardly be increased by PC IgEs, while it is robustly induced by HC IgEs, IgE+Ag or IgE+anti-IgE. Consistent with this, only HC, but not PC, IgEs could induce the activation of MAPKs and Akt (Kalesnikoff et al 2001, Kitaura et al 2003). Optimal concentrations of antigen or anti-IgE used to stimulate mast cells under IgE+Ag or IgE+anti-IgE activate these pathways, causing all the activation events. Therefore, IgE+Ag and IgE+anti-IgE generally provide stronger FceRI-aggregating stimuli than monomeric IgEs. A recent study indicated that the rate of receptor internalization serves as a surrogate indicator of stimulation intensity in IgE+Ag and IgE+antiIgE (Kitaura et al 2004).

Mechanisms of mast cell survival and activation by monomeric IgE Differences in the quantity and quality of stimulation provided by these different modes of FceRI aggregation would be key to our better understanding of what happens when FceRI is bound by IgE and what difference makes HC versus PC IgEs. As described above, HC IgEs use an autocrine and paracrine secretion of a cytokine or cytokines to promote mast cell survival. Saito and colleagues have recently identified interleukin (IL)3 as a cytokine that is at least partly responsible for this effect (Takashi Saito, personal communication). The failure of PC IgE molecules to induce the production of IL3 or other survival-promoting cytokines might be simply due to low efficiency to induce receptor aggregation. Potentially along this line, Saito and colleagues showed that the g subunit ITAM is critical for

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IgE+Ag-induced degranulation and monomeric IgE-mediated survival, but not for monomeric IgE-mediated receptor up-regulation (Sakurai et al 2004). Using CD8/FcRg chimeras, they also demonstrated that survival can be induced by weaker stimulation than that needed for degranulation and is likely mediated by sustained extracellular signal-regulated kinase (ERK) activation (Yamasaki et al 2004). A recent study by Kitaura et al (2004) also showed that weak to moderate stimulation with IgE+anti-IgE or IgE+Ag enhances survival, while stronger signals are required for degranulation and IL6 production. On the other hand, receptor stimulation induced by monomeric IgE might be qualitatively different than that by IgE+Ag or IgE+anti-IgE. Interestingly, sensitivity to Ca2+ channel blockers was shown to be different between monomeric IgE and IgE+Ag. Kitaura et al (2004) showed that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL6 production, and survival are sensitive to monovalent hapten. IgEinduced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, HC IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL6 production, and survival require Syk. Collectively, signals emanated from IgE-bound FceRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes. Model for monomeric IgE-mediated receptor aggregation Except for receptor up-regulation, the other monomeric IgE-induced events such as survival, degranulation and cytokine production seem to require proper receptor aggregation. Since these events are sensitive to monovalent hapten, the variable region of IgE seems to be involved in receptor aggregation. Interestingly, a recent study described a molecule termed p28 in rat rat basophilic leukaemia (RBL)-2H3 mast cells (Charles et al 2004). This molecule was shown to be associated with ~50% of FceRI in RBL-2H3 cells and dissociate from the receptor when the cells were incubated with IgEs. Although p28 seemed to be inadvertently recognized by a monoclonal antibody raised against phospholipid scramblase 1, its identity has not been known yet. However, assuming that p28 is generally expressed in mast cells, it is envisaged that p28 might prevent approximation of neighbouring receptors and that, upon binding to receptor molecules, the variable region of an IgE molecule might interact directly or indirectly with a neighbouring IgE molecule, forming a local aggregate of receptors (Fig. 1). The extent of receptor aggregation is apparently determined by the variable region of IgE molecules, which is probably the defining factor of the differences between HC and PC properties. HC IgEs induce more extensive receptor aggregation than PC IgEs do. Several studies showed that

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+IgE Direct interactions

Indirect interactions

FIG. 1. A model for monomeric IgE-induced FceRI aggregation. Before IgE binding, aggregation of FceRI on the surface of mast cells is prevented by p28 (or an equivalent protein). Upon monomeric IgE binding, p28 is dissociated from FceRI and receptor molecules are aggregated by direct receptor–receptor interactions or indirect interactions involving a bridging molecule. These interactions bring together neighbouring receptor molecules into aggregates.

variable regions of immunoglobulins bind to viral, bacterial and endogenous proteins. Therefore, it is not surprising that the variable region of IgE would interact with cell surface proteins or even IgE itself. This possibility remains to be tested. So do potential contributions of sugar moieties of IgE molecules to receptor aggregation. It is, however, unlikely that the affinity of IgEs to FceRI plays a major role in determining the difference between HC and PC IgEs, as a typical HC IgE, SPE-7, and a typical PC IgE, H1 DNP-e-206, have similar reported affinities, K d = ~15 nM. IgE-induced mast cell survival and clinical implications Several studies indicate the ability of IgE to enhance mast cell and basophil function in both mouse and human cells (Yamaguchi et al 1997, 1999, Yano et al 1997). Together with the enhanced cell survival, a positive feedback-loop hypothesis was proposed to explain inflammatory situations often found in atopic diseases associated with high serum IgE levels: ≠IgE Æ ≠FceRI, ≠mast cells Æ ≠antigen-, IgEand FceRI-dependent release of IL4, IL13, CCL3 and so on Æ ≠IgE (Kawakami & Galli 2002). Now, studies have been extended to human monoclonal IgEs: a preliminary study showed that human IgE can promote survival and chemokine production by human umbilical-cord-blood-derived mast cells in vitro (K. Matsuda, A.M. Piliponsky, S. Nakae, T. Kawakami, M. Tsai, S.J. Galli, unpublished results).

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The distinction between HC and PC IgEs is reminiscent of the dichotomy found in human IgE molecules in their ability to prime basophils for the stimulation with histamine-releasing factor (HRF), a cytokine produced by macrophages and platelets: basophils bound by IgEs (termed IgE+) derived from atopic patients, but not those (termed IgE-) from normal subjects, can release histamine and cytokines such as IL4 and IL13 in response to HRF (MacDonald et al 1987, Schroeder et al 1996). The structural basis for the difference between IgE+ and IgE- is not known yet. These observations together indicate the structural and functional heterogeneity among human and mouse IgE molecules. It is necessary to study the IgE heterogeneity and its relevance with atopic and other diseases. Acknowledgements We are grateful to Dr Daniel H. Conrad for his kind gift of mAb E1B3 and members of the Kawakami laboratory for providing bone marrow-derived mast cells used in this study. This study was supported by grants from the National Institutes of Health AI50209 and AI/GM38348 to T.K.

References Asai K, Kitaura J, Kawakami Y et al 2001 Regulation of mast cell survival by IgE. Immunity 14:791–800 Borkowski TA, Jouvin MH, Lin SY, Kinet JP 2001 Minimal requirements for IgE-mediated regulation of surface Fc epsilon RI. J Immunol 167:1290–1296 Charles N, Monteiro RC, Benhamou M 2004 p28, a novel IgE receptor-associated protein, is a sensor of receptor occupation by its ligand in mast cells. J Biol Chem 279:12312– 12318 Conroy MC, Adkinson NF Jr, Lichtenstein LM 1977 Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release. J. Immunol. 118:1317– 1321 Furuichi K, Rivera J, Isersky C 1985 The receptor for immunoglobulin E on rat basophilic leukemia cells: effect of ligand binding on receptor expression. Proc Natl Acad Sci USA 82:1522–1525 Hsu C, MacGlashan D Jr 1996 IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells. Immunol Lett 52:129–134 Kalesnikoff J, Huber M, Lam V et al 2001 Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 14:801–811 Kawakami T, Galli SJ 2002 Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2:773–786 Kinet JP 1999 The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology. Annu Rev Immunol 17:931–972 Kitaura J, Song J, Tsai M et al 2003 Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI. Proc Natl Acad Sci USA 100:12911–12916 Kitaura J, Xiao W, Maeda-Yamamoto M et al 2004 Early divergence of Fcepsilon receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production, and survival. J Immunol 173:4317–4323

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Kitaura J, Kinoshita T, Matsumoto M et al 2005 IgE– and IgE+ Ag-mediated mast cell migration in an autocrine/paracrine fashion. Blood 105:3222–3229 Kubo S, Matsuoka K, Taya C et al 2001 Drastic up-regulation of FceRI on mast cells is induced by IgE binding through stabilization and accumulation of FceRI on the cell surface. J Immunol 167:3427–3434 Lam V, Kalesnikoff J, Lee CW et al 2003 IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE + antigen but distinct from those used by Steel factor. Blood 102:1405–1413 MacDonald SM, Lichtenstein LM, Proud D et al 1987 Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE. J Immunol 139:506–512 Malveaux FJ, Conroy MC, Adkinson NF Jr, Lichtenstein LM 1978 IgE receptors on human basophils. Relationship to serum IgE concentration. J Clin Invest 62:176–181 Metzger H 1992 The receptor with high affinity for IgE. Immunol Rev 125:37–48 Oka T, Hori M, Tanaka A et al 2004 IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells. Am J Physiol Cell Physiol 286:C256–263 Pandey V, Mihara S, Fensome-Green A, Bolsover S, Cockcroft S 2004 Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca(2+), degranulation, and membrane ruffling in the cultured rat basophilic leukemia-2H3 mast cell line. J Immunol 172:4048–4058 Parravicini V, Gadina M, Kovarova M et al 2002 Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol 3:741–748 Quarto R, Kinet JP, Metzger H 1985 Coordinate synthesis and degradation of the alpha-, beta- and gamma- subunits of the receptor for immunoglobulin E. Mol Immunol 22:1045– 1051 Sakurai D, Yamasaki S, Arase K et al 2004 FcepsilonRIgamma-ITAM Is Differentially Required for Mast Cell Function In Vivo. J Immunol 172:2374 –2381 Schroeder JT, Lichtenstein LM, MacDonald SM 1996 An immunoglobulin E-dependent recombinant histamine-releasing factor induces interleukin-4 secretion from human basophils. J Exp Med 183:1265–1270 Stallman PJ, Aalberse RC, Bruhl PC, van Elven EH 1977 Experiments on the passive sensitization of human basophils, using quantitative immunofluorescence microscopy. Int. Arch. Allergy Appl. Immunol. 54:364 –373 Tanaka S, Takasu Y, Mikura S, Satoh N, Ichikawa A 2002 Antigen-independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow-derived mast cells. J Exp Med 196:229–235 Turner H, Kinet JP 1999 Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature 402:B24 –30 Yamada N, Matsushima H, Tagaya Y, Shimada S, Katz SI 2003 Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells. J Invest Dermatol 121:1425–1432 Yamaguchi M, Lantz CS, Oettgen HC et al 1997 IgE enhances mouse mast cell Fc(epsilon)RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgEdependent reactions. J Exp Med 185:663–672 Yamaguchi M, Sayama K, Yano K et al 1999 IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord bloodderived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release. J Immunol 162:5455–5465 Yamasaki S, Ishikawa E, Kohno M, Saito T 2004 The quantity and duration of FcRgamma signals determine mast cell degranulation and survival. Blood 103:3093–3101 Yano K, Yamaguchi M, de Mora F et al 1997 Production of macrophage inflammatory protein-1alpha by human mast cells: increased anti-IgE-dependent secretion after IgEdependent enhancement of mast cell IgE-binding ability. Lab Invest 77:185–193

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DISCUSSION Pecht: Going back to the first part of your paper and the mechanisms underlying the assumed FceRI aggregation, I have earlier made the point that in this discussion we are ignoring the fact that a highly non-physiological situation is produced by taking either the bone marrow-derived mast cells (BMMCs) or the rat basophilic leukaemia (RBL) cells which carry tens to hundreds of thousands of copies of the FceRs on their respective surface, and loading them with one and the same monoclonal IgE. As a result of having such a large number of FceRs on the cells’ surface and identical bound IgEs, we are producing a situation of exceptionally high surface IgE concentration. This would lead to aggregation even if a low level of affinity for these IgEs exists for a cell membrane component: it may lead to homoaggregation or hetero-aggregation depending on what epitope the employed IgE is having an affinity for. Please do note that we are dealing with a two dimensional concentration of these IgEs on the cells’ surface. Having them in these two dimensions means that their effective concentration is orders of magnitude higher. Therefore if the employed IgE has a low affinity for an epitope by itself or on another cell membrane component, it would lead to aggregation. Dr Kawakami’s results using SPE-7 are a clear illustration of this: adding a specific monovalent hapten is blocking the effect. This clearly implies that the variable domain of the IgE (SPE-7) is involved in the induced process. We are thus clearly dealing with a non physiological situation and may conclude that it is not the monomeric IgE which induces the reported cellular responses but rather the aggregation produced under these conditions. Kawakami: I’m not sure whether I fully understand your point. What we have seen so far is that if we use a mixture of up to eight different monoclonal Ig molecules (a mixture of highly cytokinergic and poorly cytokinergic IgEs), we show that the effect is close to the average. Pecht: This average result does make sense as it probably reflects some sort of an average degree of the induced aggregation. Furthermore the case of the SPE-7 stands out: it has been shown to exhibit affinity for a protein epitope, thioredoxin in addition to that for nitro aromatic haptens which were used for its preparation and this could be the reason for its efficacy (see James et al 2003). We have recently calculated that the affinity of the cell resident IgE could be as low as 10-3 to 10-4 M and would still induce some clustering. Thus as soon as you are moving into the two-dimensional state of the cell surface, the markedly higher effective epitope and IgE concentrations will lead even with such low affinities to aggregation (see also Schweitzer-Stenner & Pecht 2005). Rivera: I’d like to address this point from an in vivo perspective. One of the issues is whether there are (patho)physiological settings where one can obtain IgE saturation of receptors, at concentrations of IgE that are above physiological. In our

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system with the Lyn knockouts this seems to be the case. We are clearly getting increases of IgE that are well above normal levels. We see all of the mast cells in these mice are completely saturated with IgE. We get an activation of mast cells as measured by the increase in circulating histamine levels. This isn’t a direct demonstration of activation of mast cells, but it suggests that mast cells are being activated. If one starts thinking of the atopic individual, perhaps these kinds of settings exist, where it really is physiological but occurs primarily in a dysregulated state. Pecht: This isn’t a criticism; it is an interesting reflection of this nonphysiological situation. Still, in the intact animal case, we should remember that the repertoire of cell-bound IgE specificities is the whole one existing in the animal, so the probability that you will induce such aggregation is considerably lower. Stevens: We showed in 1984 that fibronectin is susceptible to a protease (presumably mMCP-5) stored in the secretory granules of interleukin (IL)3-developed mouse bone marrow-derived mast cells (mBMMCs) (DuBuske et al 1984). Thus, one interpretation of your data is that the cells are releasing a protease that degrades the fibronectin that is used to block the membrane’s pores in your transwell assay. Is fibronectin degraded in your experiments? Kawakami: No. MacDonald: We have discussed the data with the mouse HRF that I sent you which is produced in Escherichia coli. Years ago I thought that HRF indeed bound IgE. Since then, we have published data that lead us to believe that it doesn’t bind IgE. In particular, it activates not only basophils (MacDonald et al 1995) but also eosinophils, which do not have the a chain of the IgE receptor (BheekhaEscura et al 2000). It also activates T cells (Vonakis et al 2003). We have gone so far as to show that if you take the IgE plus from responder donors and sensitize the RBL cell that is transfected with the human FceRI, this cell is not activated by histamine-releasing factor (HRF), even though we do get full activation with polyclonal IgE (Wantke et al 1999). I have not been able to show HRF activation of human cultured mast cells. When I sent you the HRF I recognized that because it is produced in E. coli, it is full of endotoxin. We don’t have the problem with Tolllike receptor activation in basophils because they don’t have CD14. I don’t know the mechanism: I’ve seen your data and I believe them, but are you sure that the augmentation that you are seeing when you use HRF has nothing to do with Tolllike receptor activation on a mast cell? Kawakami: We haven’t really looked at this possibility. In experiments looking at the effects of IgE, endotoxin doesn’t have anything to do with the survival effects. The patterns of the effects are very different between lipopolysaccharide and IgE and also if we use FceRIa knockout mice we don’t see any effects with IgE. This excludes the possibility that contamination by endotoxin in IgE preparation is responsible for biological effects.

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Koyasu: When you showed the production of soluble mediators, including chemokines, by the interaction of IgE and FceR, and migration of FceRIa knockout mast cells, did you basically mix wild-type and a knockout mast cells? Kawakami: We did several experiments. I just talked about two kinds in my presentation. One is using FceRIa knockout mice in the upper well, and then incubating with wild-type mast cells together with IgE in the lower well. In this setting the knockout mast cells can move from the upper well to the lower well. Koyasu: When you talk about HC and PC IgEs, is there any genetic background that can produce more of either type? This might explain a lot of observations such as the NC mice. Kawakami: Nothing is known about this, unfortunately. We hope that in the future we’ll be able to sort out whether allergic patients tend to produce more HC IgE than PC IgE. Metzger: Going back to Juan Rivera’s example of a possible physiological role, it seems to me we have had another presentation here that is also relevant. Steve and Hans, why don’t you want to defend your results on the wild-type mice? It seems to me that this is the most physiological situation. It may be that some of the audience feel that this is a very special situation, where the IgE-mediated effect is not on the response itself, but somehow is necessary for the response. In this sense, it is not an allergic response but is the most physiological example we have had of some sort of low-level signalling being necessary. One cannot exclude the fact that this low-level signalling is due to some sort of an antibody-site-mediated aggregation. It is known for example that almost all anti-DNPs react with nucleic acids. These are present in small amounts. Isn’t this the common theme? There is something that is antibody site -mediated that is causing low-level aggregation and release of soluble factors, and this occurs at a physiological level. Galli: I couldn’t put it better than you have, Henry. This is reminiscent of the discussion that occurred concerning the effects of IgE on levels of surface expression of the FceRI receptor. There was general agreement that the phenomenon occurred in vitro, and the key question then became whether there were any circumstances in which it was relevant to the expression of mast cell function in vivo. Metzger: The original observations were clinical. Galli: That is correct. Both Larry Lichtenstein and his colleagues and Rob Aalberse and his collaborators made a very similar observation at the same time: that there was a very strong direct positive correlation between serum IgE levels and levels of expression of the FceRI receptor on blood basophils (Conroy et al 1977, Stallman et al 1977, Malveaux et al 1978). The only reservation about interpreting those observations as evidence that concentrations of IgE regulated levels of expression of the FceRI on basophils was that there was no evidence that one finding necessarily was causally related to the other. For example, a cytokine or some other factor could have been responsible both for increasing levels of IgE and for enhancing surface expression of FceRI. The observation was later made that incu-

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bation with IgE could enhance levels of surface expression of FceRI on RBL cells in vitro (Furuichi et al 1985, Quarto et al 1985). The question then became, does the ability of IgE to regulate levels of FceRI have functional significance? It was shown to be functionally relevant in vitro (Hsu et al 1996, Yamaguchi et al 1997), but with Hans Oettgen’s IgE knockout mouse, it was possible to show that in the absence of physiological levels of IgE there was only 20% of the normal level of expression of the receptor on native peritoneal mast cells (Yamaguchi et al 1997). The diminished levels of FceRI surface expression could be normalized by passively administering IgE to the IgE knockout mice, indicating that IgE can regulate levels of FceRI surface expression in vivo. Getting back to John’s and Israel’s point, there is an important question here about the underlying mechanism. Then there is another important question, about in vivo relevance, namely, does this mechanism occur in some physiological or pathological responses? Hans’ data show that there is an IgE- and FceRI-dependent mechanism that can be observed in vivo, at least for some examples of contact hypersensitivity. Oettgen: With regard to the interpretation of the studies with the IgE knockout versus wild-type mice, the readout is contact sensitivity rather than direct assessment of mast cell function. It is not a direct assessment. Cockroft: Could you define exactly what you mean by HC and PC? Kawakami: It’s not precise; it isn’t an all-or-none situation. It reflects a spectrum. The only real HC is SPE-7, although Jerry Krystal’s group mentioned that an antiEPO monoclonal IgE has a strong capability of inducing cytokines. Cockroft: So this is an operational definition. In your original publication you showed that IgE could promote survival in the absence of activation of all the normal signalling pathways. Is the survival phenomenon that you previously published separate from the ability of IgE to activate downstream signalling pathways? Kawakami: I’m glad you mentioned this, because I didn’t mention the cytokine that is responsible for IgE-induced survival. This has now been identified as IL3 by Takashi Saito’s group in Japan (Kohno et al 2005). Still, even though they used mast cells from IL3 knockout mice, they could show some survival effect. This survival effect is therefore probably due to at least two different mechanisms. One is soluble factor dependent (predominantly IL3) and the other is probably due to other cytokines. There may also be a cytokine independent mechanism. Cockroft: You talk about the release of soluble factors. Presumably they are coming from activation of the mast cells themselves. You need to invoke all the normal signalling pathways that you associate with DNP antigen ligation. When you look at your chemotaxis assays, presumably you could have done exactly the same experiment using soluble antigen. Is the IgE stimulation in parallel with the antigen DNP stimulation? Kawakami: Yes and no. There are quantitative and qualitative differences in terms of signalling. The differences are mostly quantitative, but there are some qualitative

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differences, such as the requirement for Src family kinases in some functions. Some groups have data showing that there are differences in sensitivity to calcium channel inhibitors when IgE alone is compared with IgE plus antigen. Razin: I think we put too much effort into understanding the mechanism of the effect of the monomeric IgE via aggregation which means the bridging of two receptors. Why couldn’t it be that the monomeric IgE is just bound to the receptor, like cytokines, and there is some kind of change in the micromovement of the receptor that induces the signalling? Pecht: There is no need for this, because there are by now sufficient experimental and theoretical reasons to assume that some degree of FceRI aggregation is induced by the protocols employing monomeric IgEs. Everything Toshi told us fits with this notion. The response of the cells fits it, so do his hapten inhibition results and our own calculations. Why go back to old-time models which were effectively eliminated some 30 years ago. Metzger: My own response would be that we know one mechanism by which one can activate the cell. That is, we have one model, the kinase phosphorylation model which seems to fit with other receptors. But there are a tremendously wide number of ways that receptors can activate cells. If one is going to postulate that a monomeric receptor is able to stimulate, one has to then follow this up and try to figure out what the mechanism is. On the other hand, the hapten inhibition experiment suggests that the monomeric mechanism is not the predominant one. Pecht: I agree, but there is one more point we all agree on so far: it is the fact that by loading the RBL-2H3 cells or even the BMMCs with one and the same monoclonal IgE, we are producing a completely different biophysical situation. Rivera: One of the points we haven’t discussed is the studies from Bridget Wilson’s group, using high resolution electron microscopy. One of the findings is that some receptors appear to be clustered under the resting conditions. Perhaps this is an important point here: one might find receptors that are in microclusters under resting conditions. Kawakami: By ‘resting’ do you mean IgE sensitized? Rivera: That’s correct. Pecht: These observations could indeed be interpreted in a more simplistic sense if we view the situation in the cell membrane as a static one. This, however is not the case and we should ask ourselves what is the nature of the reported aggregates: Are they sufficiently long-lived to produce a signal or just transient and caught as a snapshot by the experimental protocol of the EM experiments? Rivera: The studies by Nicolas Charles and Marc Benhamou (Charles et al 2004) are interesting with regards to the p28 protein. Do you have any data suggesting whether your HC or PC IgEs make any difference with regards to the dissociation of this protein from the receptor?

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Kawakami: I didn’t have time to mention the detail. This monoclonal antibody used by Charles and Benhamou is one of the antibodies against phospholipid scramblase 1. I collaborated with Peter Sims at the Scripps Research Institute. They have a bunch of monoclonal antibodies against the same molecule. However, none of them could identify the so-called p28. Therefore, the Charles-Benhamou antibody must have some special capacity to recognize p28. Bradding: Going back to your migration assay, have you done any controls to see whether this is really directed migration? Kawakami: We tested it using so-called checkerboard analysis, and we found that both chemotaxis and chemokinesis contribute to the movement of mast cells. This is different from the migration induced by SCF. Marshall: One of the other physiological examples of high IgE is nematode parasite infection. I was looking at the changes in mast cell numbers you reported in the skin and trying to rationalize why in nematode infection we don’t see larger changes in mast cell numbers in the skin, if elevating monomeric IgE should have that effect. Kawakami: We made a very high concentration area; a very small patch. Only this area has a steep gradient of IgE density. Marshall: So you think it is recruitment to that local area? Kawakami: Yes. Marshall: So in a systemic context this effect wouldn’t be seen. References Bheekha-Escura R, MacGlashan Jr DW, Langdon JM, MacDonald SM 2000 The human recombinant histamine releasing factor (HrHRF) activates human eosinophils and the eosinophiliclike cell line, AML 14-3D10. Blood 96:2191–2198 Charles N, Monteiro RC, Benhamou M 2004 p28, a novel IgE receptor-associated protein, is a sensor of receptor occupation by its ligand in mast cells. J Biol Chem 279:12312–12318 Conroy MC, Adkinson NF Jr, Lichtenstein LM 1977 Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release. J Immunol 118:1317–1321 DuBuske L, Austen KF, Czop J, Stevens RL 1984 Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells. J Immunol 133:1535–1541 Furuichi K, Rivera J, Isersky C 1985 The receptor for immunoglobulin E on rat basophilic leukemia cells: Effect of ligand binding on receptor expression. Proc Natl Acad Sci USA 82:1522–1525 Hsu C, MacGlashan D Jr 1996 IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells. Immunol Lett 52:129–134 James LC, Roversi P, Tawfik DS 2003 Antibody multispecificity mediated by conformational diversity. Science 299:1362–1367 Kohno M, Yamasaki S, Tybulewicz VL, Saito T 2005 Rapid and large amount of autocrine IL-3 production is responsible for mast cell survival by IgE in the absence of antigen. Blood 105:2059–2065 MacDonald SM, Rafnar T, Langdon J, Lichtenstein LM 1995 Molecular identification of an IgEdependent histamine releasing factor. Science 269:688–690

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Malveaux FJ, Conroy MC, Adkinson NF Jr, Lichtenstein LM 1978 IgE receptors on human basophils. Relationship to serum IgE concentration. J Clin Invest 62:176–181 Quarto R, Kinet J-P, Metzger H 1985 Coordinate synthesis and degradation of the alpha-, betaand gamma-subunits of the receptor for immunoglobulin E. Mol Immunol 22:1045–1051 Schweitzer-Stenner R, Pecht I 2005 Death of a dogma or enforcing the artificial: monomeric IgE binding may initiate mast cell response by inducing its receptor aggregation. J Immunol 174:4461–4464 Stallman PJ, Aalberse RC, Bruhl PC, van Elven EH 1977 Experiments on the passive sensitization of human basophils, using quantitative immunofluorescence microscopy. Int Arch Allergy Appl Immunol 54:364 –373 Vonakis BM, Sora R, Langdon JM, Casolaro V, MacDonald SM 2003 Inhibition of cytokine gene transcription by human recombinant histamine-releasing factor in human T lymphocytes. J Immunol 171:3742–3750 Wantke F, MacGlashan DW, Langdon JM, MacDonald SM 1999 The human recombinant histamine releasing factor rHRF: Functional evidence that rHRF does not bind to the IgE molecule. J Allergy Clin Immunol 103:642–648 Yamaguchi M, Lantz CS, Oettgen HC et al 1997 IgE enhances mouse mast cell FceRI expression in vitro and in vivo: Evidence for a novel amplification mechanism in IgE-dependent reactions. J Exp Med 185:663–772

RabGEF1, a negative regulator of Ras signalling, mast cell activation and skin inflammation See-Ying Tam*, Janet Kalesnikoff*, Susumu Nakae*, Mindy Tsai* and Stephen J. Galli*†1 Departments of *Pathology and †Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA

Abstract. Mast cell activation induced by the aggregation of FceRI with IgE and antigen is mediated through the activation of multiple protein kinase cascades. This process induces mast cells to undergo degranulation, to synthesize and release lipid mediators, and to secrete multiple cytokines, chemokines and growth factors. We found that RabGEF1 (Rabex-5) binds to Ras and negatively regulates Ras activation and downstream effector pathways during FceRI-dependent mouse mast cell activation. Mast cells derived from RabGEF1-deficient mice exhibit significantly enhanced levels of degranulation, release of lipid mediators and secretion of cytokines in response to FceRI aggregation. RabGEF1 knockout mice have increased perinatal mortality and the mice that do survive develop severe skin inflammation and increased numbers of mast cells in the dermis, some of which exhibit morphological evidence of degranulation. These mice also show elevated concentrations of serum histamine and IgE. Thus, RabGEF1 is a negative regulator of Ras signalling and FceRI-dependent mast cell activation in vitro, and a lack of RabGEF1 results in the development of elevated numbers of mast cells in the skin and severe skin inflammation in vivo. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 115–130

Mast cells can be activated through the aggregation of high-affinity IgE receptors (FceRI) expressed on the cell surface by the binding of multivalent antigen to FceRI-bound IgE molecules (Kinet 1999, Metzger 1992, Rivera 2002, Siraganian 2003). This FceRI-dependent mast cell activation results in degranulation, with the secretion of preformed mediators stored in the cells’ cytoplasmic granules, as well as the synthesis and release of lipid mediators and many cytokines, chemokines and growth factors (Galli 2000, Galli et al 2005, Kawakami & Galli 2002, Metcalfe et al 1997). 1

This paper was presented at the symposium by Stephen J. Galli to whom correspondence should be addressed. 115

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The synthesis and release of lipid mediators and cytokines in response to FceRIdependent mast cell activation are mediated through the activation of signalling pathways that include Ras and Ras-mediated downstream protein kinase cascades (Beaven & Baumgartner 1996, Hirasawa et al 1995a,b, Jabril-Cuenod et al 1996, Kawakami et al 2003). Ras proteins are small GTPases that are critically involved in the control of the differentiation, proliferation and function of various cell types (Katz & McCormick, 1997). Activation of Ras to the GTP-bound form, in response to ligand–receptor interactions at the cell surface, functions as a molecular switch for the activation of downstream signalling cascades. For example, activated Ras binds directly to its key effector Raf-1 to activate the ERK MAP kinase cascade. However, Ras can regulate a wide spectrum of cellular functions and multiple effectors of Ras, in addition to Raf-1, that mediate distinct biological actions of Ras have been identified (Katz & McCormick 1997, Shieldset al 2000). These Ras effectors constitute a group of structurally and functionally distinct regulatory factors that include guanine-nucleotide exchange factors (GEFs) for Ral, Ras GTPaseactivating proteins, MEK kinase 1, AF6, phosphatidylinositol-3-kinase and Rasinteraction-interference 1 (RIN1) (Katz & McCormick 1997, Shields et al 2000). The aggregation of FceRI induced by IgE and antigen binding leads to the activation of downstream signalling cascades, including the phospholipase Cg (PLCg )/protein kinase C (PKC) cascade required for degranulation, and the Ras/ERK/phospholipase A2 cascade critical for the release of cytokines and products of arachidonic acid metabolism (Beaven & Baumgartner 1996, Hirasawa et al 1995a,b, Jabril-Cuenod et al 1996, Kawakami et al 2003, Parravicini et al 2002, Siraganian 2003). To identify novel factors that can regulate FceRIdependent mast cell activation, we undertook a functional genomics approach to isolate genes that were differentially regulated in mouse mast cells after FceRIdependent activation. Identification of RabGEF1 in mast cells activated via FceRI aggregation Differential screening using the differential display technique in mouse mast cells which had been stimulated through FceRI by IgE and specific antigen revealed a gene whose expression was up-regulated at 30 min and 1 hour after antigen challenge but returned to baseline after ~2 hours (Fig. 1a) (Tam et al 2004). Subsequent cDNA cloning and sequencing showed that the gene we had isolated was an orthologue of bovine Rabex-5, a Rab5 binding protein that shows in vitro GEF activity and is designated as RabGEF1 by the Mouse Genome Database (Horiuchi et al 1997, Lippe et al 2001). Western blot analysis with an antibody against mouse RabGEF1 revealed that RabGEF1 protein expression was decreased at 1 hour and 2 hours after FceRI aggregation and returned to ~ basal levels by about 6 hours (Fig. 1b) (Tam et al 2004).

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FIG. 1. Expression of RabGEF1 in mouse mast cells activated by FceRI aggregation in vitro. (a) Northern blot analysis of RabGEF1 mRNA expression in mouse BMMCs stimulated by FceRI aggregation. BMMCs were sensitized with an anti-DNP IgE mAb (H1-DNP-e26) for 2 hours at 10 mg/ml and then challenged with DNP30–40HSA (50 mg/ml). (b) Western blot analysis with anti-mouse RabGEF1 showing RabGEF1 protein expression in Cl.MC/C57.1 (C57) mast cells and in BMMCs in response to FceRI aggregation under conditions described in (a). Reproduced with permission from Tam et al (2004).

RabGEF1 is a negative regulator of Ras signalling Mouse RabGEF1 shares significant homology in the Vps9p domain with members of a Rab5-binding protein family that includes Vps9p, RIN1 and JC265 (RIN2) (Burd et al 1996, Colicelli et al 1991). Vps9p is the GEF for the Rab5 orthologue, Vps21p, in yeast, and both RIN1 and RIN2 exhibit GEF activity for Rab5 (Hama et al 1999, Saito et al 2002, Tall et al 2001). Moreover, RIN1 and JC265 can inhibit an activated RAS2 allele in yeast (Colicelli et al 1991). Since RIN1 can negatively regulate Ras activity and certain Ras-mediated functions in vitro, we assessed whether RabGEF1 could bind to Ras and influence Ras activation. Using the yeast twohybrid system, we showed that mouse RabGEF1 can interact physically with wildtype mouse H-Ras ( Tam et al 2004). Moreover, immuoprecipitation using the Ras-binding domain of Raf-1 and lysates of mast cells stimulated with IgE and antigen showed that RabGEF1 was present in the immunoprecipitate containing activated Ras-GTP ( Tam et al 2004). To assess whether RabGEF1 can also function as a negative regulator of Rasmediated signalling responses during mast cell activation, we initially used an antisense expression approach to ‘knock down’ RabGEF1 expression. Following FceRI aggregation, mast cells transfected with the RabGEF1 antisense expression construct exhibited reduced RabGEF1 expression, but higher basal Ras activity, higher levels of Ras activation and a more sustained activation compared to those of control transfectants (Tam et al 2004). Moreover, activation of ERK, the downstream effector of the Ras/Raf-1/MEK cascade, was significantly potentiated in

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FIG. 2. Potentiation of Ras-mediated signalling responses in Rabgef1-/- BMMCs in response to FceRI-dependent cell activation. (a) Kinetics of Ras activation induced by FceRI aggregation in Rabgef1-/- (-/-) vs. wild-type (+/+) BMMCs. Cells were sensitized with anti-DNP IgE mAb (H1DNP-e26) overnight at 2 mg/ml and then challenged with DNP-HSA (10 mg/ml). (b) Kinetics of ERK activation induced by FceRI aggregation, as in (a), in Rabgef1-/- (-/-) vs. wild-type (+/+) BMCMCs. Reproduced with permission from Tam et al (2004).

the antisense transfectants after stimulation via FceRI aggregation (Tam et al 2004). These results are consistent with the conclusion that decreased RabGEF1 expression can enhance activation of Ras and Ras-mediated signalling pathways during FceRI-dependent mast cell activation. To assess the effects of a total absence of RabGEF1 expression on mast cell activation, we used gene targeting to generate Rabgef1-/- mice on either a 129/SvEv background or a mixed 129Sv/Ev and C57BL/6 background. Bone marrowderived cultured mast cells (BMMCs) derived from these Rabgef1-/- mice had slightly lower expression of FceRI compared to the corresponding wild-type BMMCs (Tam et al 2004). However, these Rabgef1-/- mast cells exhibited higher Ras activation, both at baseline and after stimulation with IgE and antigen, compared with wildtype BMMCs (Fig. 2a) (Tam et al 2004). Similarly, Rabgef1-/- BMMCs, compared with the wild-type cells, exhibited higher ERK activation at baseline and after FceRI aggregation, as well as a more sustained pattern of ERK activation after stimulation with IgE and antigen (Fig. 2b) (Tam et al 2004). Our data are thus consistent with the results of our RabGEF1 knock-down studies, indicating that RabGEF1 can function as a negative regulator of Ras-mediated signalling responses in mast cells. RabGEF1 is a negative regulator of mast cell activation Using our RabGEF1 antisense transfectants, we assessed whether ‘knocked-down’ expression of RabGEF1 can lead to changes in the characteristic functional


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responses induced by FceRI-dependent activation of mast cells. We found that there were no significant differences between the amounts of b-hexosaminidase (b-Hex), a representative preformed mediator, released from the antisense or control transfectants in response to FceRI aggregation. However, the release of interleukin 6 (IL6) and prostaglandin D2 (PGD2) was significantly enhanced in the antisensetransfected cells (Tam et al 2004). These findings thus support the conclusion that RabGEF1 can negatively regulate the release of both lipid mediators and cytokines from mast cells activated by FceRI aggregation. Consistent with the results of our knock-down studies, Rabgef1-/- BMMCs released significantly more lipid mediators (PGD2 and leukotriene C4 [LTC4]) and cytokines (IL6 and tumor necrosis factor [TNF]) in response to FceRI aggregation compared to the wild-type mast cells (Fig. 3b–e) (Tam et al 2004). These data, together with those generated from our knock-down studies, are consistent with the current view that the Ras/Raf-1/MEK/ERK cascade represents the major effector signalling pathway for the synthesis and release of lipid mediators and cytokines in mast cells.

FIG. 3. Enhanced release of mediators and cytokines in BMMCs derived from Rabgef1-/- mice (129/SvEv or mixed 129/SvEv and C57BL/6 background) in response to FceRI-dependent cell activation. Release of (a) b-Hex, (b) PGD2, (c) LTC4, (d) IL6 and (e) TNF induced by FceRI aggregation (after incubation with anti-DNP IgE mAb [H1-DNP-e26] overnight at 2 mg/ml and then challenged with various concentrations of DNP-HSA for [a–c] 1 hour or [d, e] 6 hours) in Rabgef1-/- (-/-) vs. wild-type (+/+) BMMCs. Values shown are mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 vs. wild-type controls by unpaired Student’s t-test, two-tailed. Reproduced with permission from Tam et al (2004).

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FIG. 4. Enhanced degranulation and secretion of IL6 in fetal liver-derived mast cells generated from Rabgef1-/- mice (WB ¥ C57BL/6F2 background) in response to FceRI-dependent cell activation. Release of (a) b-Hex and (b) IL6 induced by FceRI aggregation (after incubation with anti-DNP IgE mAb [H1-DNP-e26] overnight at 2 mg/ml and then [a] challenged with various concentrations of DNP-HSA for 1 hour or [b] challenged with 20 ng/ml of DNP-HSA for 6 hours) in WB ¥ C57BL/6F2 Rabgef1-/- (-/-) vs. wild-type (+/+) fetal liver-derived mast cells. Values shown are mean ± SEM. *** = P < 0.01 vs. wild-type controls by unpaired Student’s t-test, two-tailed.

However, in contrast to the results obtained in our knock-down studies, we found that the amounts of b-Hex released from Rabgef1-/- BMMCs in response to IgE and antigen stimulation were significantly enhanced relative to those in wild-type mast cells (Fig. 3a) (Tam et al 2004). A lack of RabGEF1 was also associated with enhanced release of b-Hex and IL6 in fetal liver-derived mast cells that had been derived from Rabgef1-/- mice generated on a WB/Re X C57BL/6F2 background (Fig. 4a,b). These results thus show that RabGEF1 can negatively regulate degranulation and the release of granule-associated mediators in mouse mast cells derived from different genetic backgrounds. Stem cell factor (SCF, also known as Kit ligand), the ligand for the c-Kit receptor and the major mast cell survival and growth factor, can also induce mast cell activation, resulting in the release of cytokines such as IL6 (Gagari et al 1997, Galli et al 1994, Wershil et al 1992). We found that SCF can induce the up-regulation of RabGEF1 mRNA expression in mouse mast cells, thus suggesting a role for RabGEF1 in c-Kit-dependent signalling in mast cells (our unpublished data). Furthermore, BMMCs derived from Rabgef1-/- mice exhibited enhanced activation of Ras and ERK and increased IL6 release compared to wild-type BMMCs in response to SCF stimulation (our unpublished data). These studies indicate that RabGEF1 can also function as a negative regulator of c-Kit-dependent mast cell activation.


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RabGEF1 is a negative regulator of skin inflammation and mast cell development in vivo All Rabgef1-/- pups of the WB/Re X C57BL/6F2 background died within 1 day of birth. Many Rabgef1-/- pups of the 129/SvEV or mixed 129/SvEv and C57BL/6 background also died, generally during the first week of life, and those few which survived to adulthood developed severe skin inflammation. The skin lesions were detectable microscopically in Rabgef1-/- mice at 3–4 weeks of age and were grossly observable at 6–8 weeks. Grossly, the skin lesions appeared as raised, white plaquelike scales affecting the ear pinnae and the periocular skin (associated with facial alopecia), which eventually spread to involve the skin of the cervical area and dorsum. Histologically, the skin lesions exhibited epidermal hyperplasia, with hyperkeratosis, as well as prominent dermal inflammation accompanied by increased vascularity (Tam et al 2004). In severe lesions, foci of intracellular oedema in the epidermis and epidermal microabscesses, consisting of neutrophil accumulation in the epidermis, were also found. Dense infiltrates of lymphocytes, eosinophils and monocytes/macrophages, as well as many mast cells, were present in the dermis (Tam et al 2004). Rabgef1-/- mice also exhibited serum concentrations of IgE and histamine that were significantly higher than those in the wild-type mice (Tam et al 2004). The skin of Rabgef1-/- mice exhibited significantly higher numbers of mast cells in the dermis than did the skin of the wild-type mice (Tam et al 2004). This difference was apparent in mice as young as 1 week of age, even before grossly obvious lesions appeared in the skin. In the adult Rabgef1-/- mice, mast cell numbers were significantly higher at sites showing severe inflammation than in less severely affected areas, and some mast cells showed histological evidence of degranulation (Tam et al 2004).

Conclusions We demonstrated that RabGEF1 can interact physically with Ras and can negatively regulate Ras-mediated signalling and functional responses during FceRI-dependent or c-Kit-dependent mouse mast cell activation. RabGEF1-deficient mice exhibit perinatal mortality, severe skin inflammation and increased numbers of mast cells in the dermis, as well as increased blood concentrations of IgE and histamine. RabGEF1 was initially identified as a binding protein and GEF for Rab5. The negative regulatory effects of RabGEF1 on GTP loading appear to be specific for Ras, since both our antisense experiments and studies of Rabgef1-/- mast cells indicated that RabGEF1 does not negatively regulate activation of the Rho family of GTPases such as Rac or Cdc42 (Tam et al 2004).

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BMMCs derived from Rabgef1-/- mice exhibited activation of Ras and ERK under baseline conditions in the absence of stimulation with known specific antigen, as well as enhanced and more sustained activation of Ras and ERK after FceRI aggregation or c-Kit receptor activation. The constitutively activated state of Ras in these Rabgef1-/- mast cells is reminiscent of that observed with Nf1+/BMMCs (Ingram et al 2001). Indeed, it is of interest that neurofibromin, the protein encoded by the Nf1 tumour suppressor gene, is also a negative effector of Ras (Shieldset al 2000). Furthermore, the enhanced basal Ras and ERK activation observed with the Rabgef1-/- mast cells may explain, in part, our observations that these cells exhibited increased degranulation, in addition to the expected enhancement in the release of cytokine and lipid mediators, upon FceRI aggregation. Recent reports have suggested that functionally significant overlap exists between the PLCg/PKC cascade and the Ras/ERK/PLA2 cascade in the orchestration of mast cell activation responses (Beaven & Baumgartner 1996, Hirasawa et al 1995a,b, Jabril-Cuenod et al 1996, Kawakami et al 2003, Parravicini et al 2002, Siraganian 2003). In Rabgef1-/- BMMCs, the constitutive basal ERK activation, and the even higher and more sustained ERK activation after stimulation with IgE and antigen, perhaps can promote interactions between the two pathways and thereby lead to enhanced degranulation, as well as the enhanced release of lipid mediators and cytokines. We envision several potentially important areas for future research. RabGEF1 was first identified as a binding protein and GEF for Rab5, a small GTPase involved in early endosomal trafficking and endocytosis (Horiuchi et al 1997, Lippe et al 2001). The Rabgef1-/- mice, and the mast cells and other cell lineages that can be derived from these animals, may be useful in attempts to define further the roles of RabGEF1 in vesicular trafficking and related cellular functions. Given the wide tissue distribution of RabGEF1 mRNA expression (our unpublished data), we think it is likely that the effects of RabGEF1 deficiency in mast cells contribute to some, but not all, aspects of the pathology observed in the Rabgef1-/- mice. It will be important to elucidate further the mechanisms by which a lack of RabGEF1 results in the various phenotypic abnormalities in Rabgef1-/mice, as well as the abnormalities in mast cells and the other cell lineages that are affected by a lack of this protein. Notably, at least 178 single-nucleotide polymorphisms in the Rabgef1 gene have so far been identified in humans (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=snp). Thus, it will be of interest to determine, both in mice and in humans, whether specific mutations or polymorphisms affecting RabGEF1 structure/function can have significant consequences in the affected individuals. Finally, it will be of interest to assess whether RabGEF1’s ability to function as a significant negative regulator of the signalling and functional responses elicited during mast cell activation can be manipulated to achieve therapeutic ends, for


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example in those diseases, such as asthma and atopic dermatitis, that are associated with IgE- and antigen-dependent mast cell activation. Acknowledgements This work was funded by grants from US Public Health Service (AI-23990, CA-72074 and HL67674 to S.J.G.).

References Beaven MA, Baumgartner RA 1996 Downstream signals initiated in mast cells by FceRI and other receptors. Curr Opin Immunol 8:766–772 Burd CG, Mustol PA, Schu PV, Emr SD 1996 A yeast protein related to a mammalian Ras-binding protein Vps9p is required for localization of vacuolar proteins. Mol Cell Biol 16:2369–2377 Colicelli J, Nicolette C, Birchmeier C, Rodgers L, Riggs M, Wigler M 1991 Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 88:2913–2917 Gagari E, Tsai M, Lantz CS, Fox LG, Galli SJ 1997 Differential release of mast cell interleukin6 via c-kit. Blood 89:2654 –2663 Galli SJ 2000 Mast cells and basophils. Curr Opin Hematol 7:32–39 Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CMM, Tsai M 2005 Mast cells as “tunable” effector and immunoregulatory cells: Recent advances. Annu Rev Immunol 23:749–786 Galli SJ, Zsebo KM, Geissler EN 1994 The kit ligand stem cell factor. Adv Immunol 55:1–96 Hama H, Tall GG, Horazdovsky BF 1999 Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport. J Biol Chem 274:15284–15291 Hirasawa N, Santini F, Beaven MA 1995a Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line. Indications of different pathways for release of arachidonic acid and secretory granules. J Immunol 154:5391–5402 Hirasawa N, Scharenberg A, Yamamura H, Beaven MA, Kinet JP 1995b A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by FceR1 is not shared by a G protein-coupled receptor. J Biol Chem 270:10960–10967 Horiuchi H, Lippe R, McBride HM et al 1997 A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90:1149–1159 Ingram DA, Hiatt K, King AJ et al 2001 Hyperactivation of p21(ras) and the hematopoieticspecific Rho GTPase Rac2 cooperate to alter the proliferation of neurofibromin-deficient mast cells in vivo and in vitro. J Exp Med 194:57–69 Jabril-Cuenod B, Zhang C, Scharenberg AM et al 1996 Syk-dependent phosphorylation of Shc. A potential link between FceRI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2. J Biol Chem 271:16268–16272 Katz ME, McCormick F 1997 Signal transduction from multiple Ras effectors. Curr Opin Genet Dev 7:75–79 Kawakami T, Galli SJ 2002 Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2:773–786 Kawakami Y, Kitaura J, Yao L et al 2003 A Ras activation pathway dependent on Syk phosphorylation of protein kinase C. Proc Natl Acad Sci USA 100:9470–9475 Kinet JP 1999 The high-affinity IgE receptor (FceRI): from physiology to pathology. Annu Rev Immunol 17:931–972

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Lippe R, Miaczynska M, Rybin V, Runge A, Zerial M 2001 Functional synergy between Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a complex. Mol Biol Cell 12:2219–2228 Metcalfe DD, Baram D, Mekori YA 1997 Mast cells. Physiol Rev 77:1033–1079 Metzger H 1992 The receptor with high affinity for IgE. Immunol Rev 125:37–48 Parravicini V, Gadina M, Kovarova M et al 2002 Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol 3:741–748 Rivera J 2002 Molecular adapters in FceRI signaling and the allergic response. Curr Opin Immunol 14:688–693 Saito K, Murai J, Kajiho H, Kontani K, Kurosu H, Katada T 2002 A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5. J Biol Chem 277:3412–3418 Shields JM, Pruitt K, McFall A, Shaub A, Der CJ 2000 Understanding Ras: ‘it ain’t over ‘til it’s over’. Trends Cell Biol 10:147–154 Siraganian RP 2003 Mast cell signal transduction from the high-affinity IgE receptor. Curr Opin Immunol 15:639–646 Tall GG, Barbieri MA, Stahl PD, Horazdovsky BF 2001 Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev Cell 1:73–82 Tam SY, Tsai M, Snouwaert JN, Kalesnikoff J, Scherrer D, Nakae S, Chatterjea D, Bouley DM, Galli SJ 2004 RabGEF1 is a negative regulator of mast cell activation and skin inflammation. Nat Immunol 5:844–852 Wershil BK, Tsai M, Geissler EN, Zsebo KM, Galli SJ 1992 The rat c-kit ligand stem cell factor induces c-kit receptor-dependent mouse mast cell activation in vivo. Evidence that signaling through the c-kit receptor can induce expression of cellular function. J Exp Med 175:245–255

DISCUSSION Austen: Do you feel that the mast cell defects you see in the skin of the knockout animal are the consequence of a direct mast cell effect of the knockout? In other words, how selective is the distribution of this molecule to various cell types? Galli: We have done some in situ hybridization studies that show that RabGEF1 mRNA is highly expressed in the CNS and epithelial cells of the gastrointestinal (GI) tract and other sites, as well as in mast cells. Based on our in vitro data, which show activation of Ras and ERK at ‘baseline’ in RabGEF1 knockout mast cells, it is possible that this alone can explain why the RabGEF1 knockout mast cells also appear to be activated in vivo. While we have no direct evidence for activated Ras in the mast cells of RabGEF1 knockout mice in vivo, this could explain why mast cells appear activated in these animals. However, my guess is that there probably is more to it than that, and we certainly haven’t ruled out some potential effects, on Rab guanine nucleotide exchange factor (GEF) 1 knockout mast cells, of the lack of RabGEF1 in some other cell type(s). Rivera: We all appreciate that IgE is the most important immunoglobulin! I was wondering whether you have any data on other immunoglobulins? Might this also be a B cell phenotype? Galli: That is an excellent point. I can’t think of a cell in which Ras activation doesn’t play a role in some part of its biology. There do appear to be other immuno-


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logical abnormalities in this animal (e.g. IgG1 levels are also elevated), but we are not ready to draw firm conclusions until we do more work in this area. Oettgen: Is the skin phenotype restricted to hyperkeratosis and increased mast cell numbers, or is there also inflammation? Are there eosinophils in the skin? Galli: The skin lesions are associated with an extensive inflammatory infiltrate. These infiltrates contain many eosinophils, but the most prevalent of the inflammatory cells in the lesions that we have examined so far are lymphocytes and neutrophils. Brown: Are you able to derive WBB6(F1) mast cells from these knockout mice? Galli: We generated a WBB6F1 embryonic stem (ES) cell line and then we targeted the gene in these ES cells. We can generate mast cells from such ES cells in vitro, and can also generate mast cells in vitro from the fetal liver cells of the Rabgef1-/- mice which we generated on the WBB6 background. Brown: Have you done any transfer experiments into W/W v mice? Galli: That’s a good point, Melissa. One of the reasons we wanted to produce these animals was to generate W/W v, Rabgef1-/- mice in order to assess whether the phenotypic abnormalities would be different if mast cells weren’t present. This work is inconclusive at present because of the severe phenotype exhibited by Rabgef1-/mice on the WBB6 background, with no pups surviving more than 12 hours after birth. I am surprised that no one has asked about the phenotype of the Rabgef1+/- mast cells, in which we find markedly reduced levels of RabGEF1 protein Brown: That was my next question! Galli: Sometimes the Rabgef1+/- mast cells give normal mediator release in response to stimulation with IgE and specific antigen, and sometimes we see enhanced release, but usually not as much as in the corresponding Rabgef1-/- mast cells. It seems that one has to reduce RabGEF1 protein levels markedly to have a significant effect on that aspect of mast cell function. Brown: Do those mast cells grow long-term in culture, like wild-type? Galli: Yes. They grow more slowly, however. Koffer: These cells exhibit down-regulated Rac and Cdc42. Did you look at their morphology? Do they ruffle? Galli: That is an excellent question, and we haven’t addressed it. Lee: Have you done engraftment of your bone marrow-derived mast cells (BMMCs) into W/W v mice? Galli: Yes. So far this has been done just once. It appears that despite the defect in proliferation that we observe in Rabgef1-/- fetal liver-derived mast cells in vitro, there is enhanced survival of these cells in the peritoneal cavity of W/W v mice in vivo. Cockcroft: In your knockout mice you find that b-hexosaminidase release is enhanced. Yet when we draw our cartoons the Ras-MAPK pathway is not required for degranulation. Why are you getting enhanced degranulation?

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Galli: There are at least two possibilities. One is that there is a connection between the Ras/Raf/ERK pathway, which is inhibited by RabGEF1, and the pathway that leads to degranulation (Xu et al 1999, Kawakami et al 2003). The second is that the ability of RabGEF1 to inhibit degranulation reflects some alternative function of RabGEF1. Two functions have already been described for RabGEF1, one in vesicular trafficking and one in Ras inhibition. There may be others. The ability of RabGEF1 to inhibit degranulation might reflect the operation of some (or some combination) of the functions of the molecule other than its ability to inhibit Ras. We are playing with the different domains of RabGEF1 to see whether alterations in such domains will differentially affect the different phenotypic characteristics of the knockout cells. We don’t have the answer to that yet. However, we consistently have observed that the RabGEF1 antisense transfectants almost never exhibit enhanced release of b-hexosaminidase, whereas the Rabgef1-/- mast cells almost always do. Cockcroft: Is there a possibility that in your antisense knockdowns, because they are only for a short time, you are not affecting expression of other genes, whereas in your knockout mouse you are? Have you done some analysis of other affected genes? Galli: This is a very good point. We have done some microarray studies to address this question, but the results aren’t ready for prime time. Is there a developmental consequence of the absence of RabGEF1, in addition to the acute effects? Certainly there are multiple phenotypic abnormalities in the mouse, including increased perinatal death. This suggests that there may be some significant effects of lacking RabGEF1 that occur during development. Whether such developmental effects influence the striking phenotype we observe in the function of Rabgef1-/- mast cells is another question. Koyasu: What is the phenotype in the gastrointestinal tract? I’m guessing that there will be an increased number of mast cells as well. Galli: Normally the number of mast cells in the small intestines of wild type mice is quite low. We haven’t seen a significant difference, so far, in the numbers of mast cells at that site in the -/- vs. +/+ mice. Koyasu: Why do the knockout mice die? Galli: We don’t know. There is no obvious gross abnormality. We are working with a veterinary pathologist to try to figure this out. Stevens: Our studies suggest that the GEF RasGRP4 stimulates prostaglandin D2 (PGD2) expression by regulating the expression of its synthase. RabGEF1 promotes the inactivation of Ras in contrast to Ras guanyl releasing protein 4 (RasGRP4) which activates Ras. Thus, did you see increased PGD2 expression in your RabGEF1-null mice? Galli: The release of PGD2 upon stimulation of the mast cells with IgE and specific antigen was also significantly enhanced when RabGEF1 was missing, either as a result of antisense treatment or in Rabgef1-/- mast cells.


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Stevens: The fact that the mast cells generated from RabGEF1-null mice produce more PGD2 than the mast cells generated from wild-type mice raises the possibility that RabGEF1 is regulating the expression of PGD2 synthase. Do the mast cells generated from RabGEF1-null have higher levels of the synthase relative to mast cells generated from wild-type mice? Galli: We have not examined that yet. Austen: Your percentage increases weren’t as high in the icosanoid mediators as they were in the cytokines. They were dramatically different. Galli: That is also true regarding the release of b-hexosaminidase: while there was a significant enhancement of IgE- and specific antigen-induced release of lipid mediators and b-hexosaminidase in Rabgef1-/- mast cells, the effect on release of the cytokines we evaluated was substantially greater. Stevens: RasGRP4-expressing human mast cell line 1 (HMC-1) cells store more total tryptase in their granules than RasGRP4-defective HMC-1 cells. The mouse orthologue of human tryptase b1 is mouse mast cell protease-6 (mMCP-6). Thus, do RabGEF1-deficient mouse mast cells contain more mMCP-6 than RabGEF1sufficient mouse mast cells? Galli: We haven’t examined that yet either. Razin: I have a question for both Steve Galli and Juan Rivera on their knock out mice. Both of you show a high level of IgE and histamine in the blood. On the single-cell level is there some kind of chronic release of histamine? If this is the case, how can the cell survive when it continuously secretes something? I would like to postulate that the histamine is derived from other cells, somewhere else. Austen: People with systemic mastocytosis may be leaking these mediators all the time. It is possible that years later having chronically induced mediators will change these mice in terms of fibrosis or tumours. Galli: These Rabgef1-/- mice ordinarily don’t live beyond about 12 weeks. They either die or we have to sacrifice them because of the severity of their skin lesions. Rivera: The Lyn-/- mice do live for an extended period. But we have to take into consideration that the levels of serum histamine that we are seeing are exponentially lower than if we gave them a systemic challenge. We are seeing some mast cells being activated but it is at a very low level, relative to giving the mice a systemic challenge. There’s an interesting phenotype that looks very similar to the phenotype you see in terms of the skin inflammation: this is when IL4 is targeted specifically to the epidermis as a transgene. These mice have the same kind of phenotype (Chan 2001). Galli: Are you referring to Bob Tepper’s work? Oettgen: Tepper’s weren’t under the K14 promoter. Larry Chan has them under the K14. Bob Tepper’s IL4 transgenic mice in which IL4 is driven by m-heavy chain enhancer and promoter have the exact same look.

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Galli: The IL4 transgenic mice described by Bob Tepper and colleagues (Tepper et al 1990) have mast cells that appear, by light and electron microscopic criteria, to undergo activation ‘spontaneously’ (Dvorak et al 1994). I would propose that the notion that was popular some time ago, that mast cells are either ‘on’ or ‘off ’, has to be abandoned now. Mast cells can exhibit a spectrum of functional activation extending from essentially none to the extensive acute activation observed during systemic anaphylaxis (Galli et al 2005). In between these two ends of the spectrum, a lot of interesting things are happening. The mast cells in these Rabgef1-/- animals are, at ‘baseline’, positioned toward the ‘active’ end of the spectrum in comparison to the mast cells in the corresponding wild-type mice. This gets to the point of whether any of the RabGEF1 SNPs that have been identified in humans are going to be of interest. If you lose a little RabGEF1 function, will your mast cells then be ‘set’, at ‘baseline’, at a level that makes them ‘hyperresponsive’ or that permits them to secrete mediators in the absence of other stimuli? Kitamura: Have you injected directly the cultured mast cells from the knockout mouse into the W/W v skin? Galli: Not yet, only into the peritoneal cavity. Kitamura: In your previous work, you showed that the skin lesion developed at the injection site of normal cultured mast cells after some treatments. There is a possibility that the skin lesion may develop spontaneously at the injection site of mutant cultured mast cells. Galli: That is an excellent experiment and it is on our list of things to do. Ono: You mentioned a single nucleotide polymorphism (SNP) analysis. Are you doing this for some sort of linkage with any of the mouse models of allergic inflammation that have been published to date? Galli: There is no linkage that we can find using the usual databases. Stevens: I agree that a SNP analysis of this gene probably would give important new information. However, different MITF and RasGRP4 isoforms have been identified in mast cells that are the result of differential splicing of the precursor transcript. Thus, have different splice variants of RabGEF1 been identified? Galli: We haven’t detected any evidence for that in either the databases or our own work. We haven’t given up, though: in the testis we found a small hybridizing transcript, but we have not yet analysed it. Marshall: In the context of these mice where you see the increased serum histamine, there is an assumption you are getting a mature mast cell and then it is becoming activated. Another explanation is that they are just failing to granulate properly and aren’t yet secreting granule products on a more persistent basis. Galli: That is a possibility, and another one was raised by Ehud Razin. There are other sources of histamine in the mouse, including the CNS. This protein is expressed in the CNS and it is conceivable that some of the histamine is coming from a non-mast cell source or sources. We don’t know enough about this yet. We


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have one possible explanation, which is the activated mast cells as a source, but this isn’t the only one. Rivera: In looking at granule structure in BMMCs from Lyn-deficient mice we don’t see any abnormalities in terms of the integrity or histochemical staining of the granules themselves. They look to be fully populated at this stage. I don’t think that we are seeing constitutive secretion per se as the mast cells mature. If we stimulate the cells with non-pharmacological agents, then they behave very much as if one is emptying out the granule contents equivalently to wild type cells. It seems that the cell is fully loaded upon release. Stevens: For those of you who intend to evaluate protease expression in the mast cells of your transgenic animals, you need to be aware of the importance of the strain’s genetic background. Mouse mast cells express varied combinations of at least 16 neutral proteases (designated as mMCPs 1–11, granzyme B, cathepsin G, carboxypeptidase A3, neuropsin and transmembrane tryptase [mTMT]/tryptase g). Some of these proteases (e.g. mMCP-2, mMCP-4, mMCP-7 and mTMT) are expressed in a strain-dependent manner (Stevens et al 1994, Hunt et al 1996, Wong et al 1999). For example, if your transgenic mouse is on a C57BL/6 mouse genetic background, the resulting mast cells will not express mMCP-7 due to a point mutation in the gene’s exon 2/intron 2 splice site. In contrast, if your transgenic mouse is on a BALB/c mouse genetic background, the resulting mast cells will express very little, if any, mTMT. MacGlashan: Have you done any studies of G protein-induced secretion on these cells? Galli: No. We have a really long list of things to do! Also, there’s a certain amount of negotiation with the postdocs about what the priorities are. One of the things that is of great interest now is figuring out why, in Rabgef1-/- mast cells which exhibit enhanced signalling with stem cell factor (SCF), there is a reduced proliferative response to SCF but with an apparently enhanced survival in vivo. It’s a complex system but an interesting one. References Chan LS, Robinson N, Xu L 2001 Expression of interleukin-4 in the epidermis of transgenic mice results in a pruritic inflammatory skin disease: an experimental animal model to study atopic dermatitis. J Invest Dermatol 117:977–983 Dvorak AM, Tepper RI, Weller PF et al 1994 Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry. Blood 83: 3600–3612 Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CMM, Tsai M 2005 Mast cells as ‘tunable’ effector and immunoregulatory cells: Recent advances. Annu Rev Immunol 23:749–786 Hunt JE, Stevens RL, Austen KF, Zhang J, Xia Z, Ghildyal N 1996 Natural disruption of the mouse mast cell protease 7 gene in the C57BL/6 mouse. J Biol Chem 271:2851–2855

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Stevens RL, Friend DS, McNeil HP, Schiller V, Ghildyal N, Austen KF 1994 Strain-specific and tissue-specific expression of mouse mast cell secretory granule proteases. Proc Natl Acad Sci USA 91:128–132 Tepper RI, Levinson DA, Stanger BZ, Campos-Torres J, Abbas AK, Leder P 1990 IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell 62:457– 467 Wong GW, Tang Y, Feyfant E et al 1999 Identification of a new member of the tryptase family of mouse and human mast cell proteases which possesses a novel COOH-terminal hydrophobic extension. J Biol Chem 274:30784 –30793

Role of CC chemokines and their receptors in multiple aspects of mast cell biology: comparative protein profiling of FceRI- and/or CCR1-engaged mast cells using protein chip technology Masako Toda, Takao Nakamura, Masaharu Ohbayashi, Yoshifumi Ikeda, Maria Dawson, Ricardo Micheler Richardson†, Andrew Alban‡, Benjamin Leed‡, Dai Miyazaki§ and Santa Jeremy Ono1 Department of Immunology, Institutes of Ophthalmology and Child Health, University College London, London EC1V 9EL, UK, † Department of Biochemistry, Meharry Medical College, Nashville TN37208, USA, ‡ Ciphergen Biosystems Ltd, Surrey GU2 7RE, UK, and § Division of Ophthalmology and Visual Science, Tottori University Faculty of Medicine, Yonago, Tottori 683-8504, Japan Abstract. Apart from the FceRI-mediated mechanism, mast cells are activated by chemokines. Evidence has accumulated indicating that there is cross-talk between the FceRI-mediated signalling pathway and CC chemokine receptor (CCR)-mediated signalling pathways in mast cells. We have found that costimulation with IgE/antigen and CC chemokine ligand 3 (CCL3) enhances degranulation but inhibits chemotaxis of rat basophilic leukaemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells). We hypothesize that this signalling cross-talk in mast cells may play important roles in the orchestration and focusing of the allergic response. In this study, we have sought information about global protein networks either enhanced or inhibited following cross-talk between the FceRI-mediated and CCR-mediated signalling pathways in mast cells. We believe this information may be useful for providing an understanding of mast cell function and in the establishment of new anti-inflammatory drugs for allergic diseases. Proteomics is a promising tool for studying protein profiles within biological samples and facilitates an understanding of the complex responses of an organism to a stimulus. Here, we show comparative data of protein profiles derived from FceRI-engaged and/or CCR1engaged RBL-CCR1 cells using protein chip array technology, a proteomic technology. We also discuss our view of the role of CC chemokines and CCRs in regulating multiple aspects of mast cell biology. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 131–144 1

This paper was presented at the symposium by Santa Jeremy Ono to whom correspondence should be addressed. 131

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Mast cells play an important role in IgE-associated allergic disorders and immune responses to parasites. FceRI cross-linking is a key event in the activation of mast cells. FceRI-mediated signalling induces a variety of events such as degranulation, increased gene transcription and cytoskeletal rearrangement in mast cells. Apart from the classical FceRI-mediated mechanism, mast cells are also activated by other substances such as chemokines and histamine-releasing factors. Chemokines are a superfamily of small, structurally related cytokine molecules characterized by their ability to affect trafficking of immune cells. There are four subclasses of chemokines (CC, CXC, C, and CXC3C), based on location of the first two cysteines in their sequence. The chemokines interact with a reciprocal family of heteotrimeric seven-transmembrane G protein-coupled receptors. Direct analysis of murine or human mast cells derived from various tissues indicates that both have the capacity to express CC chemokine receptor (CCR) 1, CCR2, CCR3, CCR4, and CCR5, and CXC chemokine receptor (CXCR) 1, CXCR2 and CXCR4, depending on in vitro differentiation scheme or tissue localization (Ochi et al 1999, de Paulis et al 2001, Ono et al 2003). The role of CCL11 and CCR3 on activation and development of mast cells The use of CC chemokine- or CCR-deficient mice has yielded important information regarding their potential roles in regulating multiple aspects of mast cells. Our study using CCL11 (eotaxin-1, a ligand of CCR3)-deficient mice showed decreased immediate hypersensitivity responses and IgE-mediated activation of conjunctival mast cells, although the CCL11 deficiency did not affect the number of the mast cells in conjunctiva (Miyazaki et al 2004). We also observed that a CCR3 antagonist inhibited degranulation of conjunctival mast cells (early phase reaction) as well as eosinophilia (late phase reaction) in a mouse model of allergic conjunctivitis (Nakamura et al 2004). These results suggest that (1) interaction of CCL11 with CCR3 is essential to prime conjunctival mast cells for FceRI-mediated activation, and (2) CCR3 antagonist has potential for treatment of allergic diseases. Interestingly, a study using CCR3-deficient mice showed an increase in baseline tracheal intraepithelial mast cells that became even more marked following allergen challenge (Humbles et al 2002). However, the increased number of mast cells was not observed in the small intestine, skin, or spleen of the allergen-challenged CCR3-deficient mice (Humbles et al 2002, Ma et al 2002). CCR3 appears to play a pivotal role for regulating development, migration and activation of mast cells in some tissues. The role of CCL3 and CCR1 in activation and migration of mast cells Apart from the interaction of CCL11 with CCR3, an interaction of CCL3 with CCR1 and/or CCR5 seems to be important in mast cell activation. Our study using

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FIG. 1. Effect of costimulation with IgE/Ag and CCL3 on degranulation and chemotaxis of RBL-CCR1 cells. RBL-CCR1 cells were sensitized with anti-DNP IgE mAb overnight. After washing, the cells were stimulated with DNP-HSA (Ag) and/or r-human CCL3 for degranulation assay (A), or applied into transwell plate for chemotaxis assay (B).

CCL3-deficient mice showed decreased degranulation of conjunctival mast cells after topical challenge of allergen to the eyes, compared with wild-type mice (Miyazaki et al 2004). The results indicate that CCL3 is essential for optimal activation of mast cells. Laffargue et al (2001) have shown that CCL3, or CCL5 significantly enhanced FceRI-mediated degranulation via PI3Kg-mediated signalling pathway in bone marrow-derived murine mast cells. We have also observed that simultaneous stimulation with CCL3 and Ag synergistically enhanced degranulation in rat basophilic leukaemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1) (Fig. 1A) and expressing human CCR5 (Toda et al 2004). The results clearly indicate that CCL3 is a costimulator for FceRI-mediated mast cell activation. In contrast with the positive effect on degranulation, the simultaneous stimulation with CCL3 and IgE/antigen affected chemotaxis of the RBL-CCR1 cells adversely. The chemotaxis toward CCL3 was decreased when RBL-CCR1 cells were sensitized with anti-DNP IgE mAb and costimulated with dinitrophenyl (DNP)HSA (Ag) and CCL3 (Fig. 1B). Small GTP-binding proteins of the Rho family, Rac, Cdc42 and Rho, control chemotaxis by mediating the reorganization of the actin cytoskeleton. The costimulation with Ag and CCL3 enhanced Rac and Cdc42

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activation, but decreased Rho kinase (ROCK), suggesting that reciprocal imbalance between small GTP-binding proteins of the Rho family result in the inhibition of chemotaxis of RBL-CCR1 cells (Toda et al 2004). Taub et al (1995) have also shown that co-engagement via FceRI and CCRs or CXCRs affected chemotaxis of bone marrow derived murine mast cells, both positively and adversely. These observations indicate that the cross-talk between the FceRI-mediated and CCR-mediated signalling pathways affects not only degranulation but also chemotaxis and many other events in mast cells. In allergic inflammatory tissues, abundant expression of chemokines such as CCL3, CCL5 and/or CCL11 have been observed both in the acute and late-phase reactions of mice and humans (Ono et al 2003). It is therefore very likely that FceRI and chemokine receptor engagement either occurs simultaneously or in relatively rapid succession on mast cells in vivo. The cross-talk between the CCR- and FceRI-mediated signalling pathways in mast cells appears to be important in the orchestration and focusing of the allergic response. Information about global protein network of the cross-talk in mast cells would advance understanding of signal transduction and function of mast cells. To examine the protein network between these two signalling pathways in mast cells we started proteomic work using RBL-CCR1 cells. Protein profile of CCR1-engaged and/or FceRI-engaged mast cells using protein chip Array technology Proteomics is a promising new tool for studying the protein profile of a biological sample in one set of experiments. The approach has the potential to reveal the complex responses of an organism to a stimulus. For a number of years, twodimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by protein identification using Mass Spectrometry has been the primary technique in conventional proteomic analyses. The technique is suited for direct comparisons of protein expression and has been used to identify proteins that are differentially expressed between samples. Despite its utility, however, it has several disadvantages, including the fact that it is laborious and requires a large amount of protein as a starting material. Protein chip array technology is a suite of analytical tools, which allows the researcher to do the rapid identification of proteomic patterns with just a small amount of material (Fung et al 2001, Hutchens & Yip 1993). After sample application onto aluminium-based protein chip, energy-absorbing molecules are added to permit desorption and ionization of specifically bound proteins. The proteins from different biological samples are effectively eluted from the protein chips by means of the surface-enhanced laser desorption/ionization (SELDI). Ionized proteins are detected, and their masses are determined by means of time-of-flight mass spectrometry.


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Ciphergen Biosystems Inc. is a pioneer in the technology. Proteomics approaches using their Protein Chip Array technology have already achieved significant successes in the study of complex diseases such as AIDS (Sun et al 2004) and cancer (Wiesner 2004). In this study, using the Protein Chip Array technology we compared the protein profile derived from FceRI-engaged and/or CCR1-engaged RBL-CCR1 cells.

Experimental procedures Sample preparation RBL-CCR1 cells (Richardson et al 2000) were sensitized with 10 ng/ml of IgE (SPE7; Sigma-Aldrich Company Ltd.) overnight and stimulated with 1 ng/ml of CCL3 (R&D systems Europe Ltd.) and/or 10 ng/ml of DNP-HSA (Ag, Sigma-Aldrich Company Ltd.) for 1 min or 6 h. The cells were then resuspended in lysis buffer containing 8 M urea, 4% CHAPS, 65 mM DTE, 40 mM Tris and protease inhibitor cocktail, Complete (Roche Diagnostics Ltd.), for 15 min at room temperature. Whole cell lysates were obtained by centrifugation at 13 000 rpm. SELDI protein profiling Samples were processed for SELDI analysis using CM10 array (an array incorporated a carboxylate chemistry that acts as a weak cation exchanger), or IMAC3 array (an immobilized metal affinity capture array with a nitriloacetic acid surface) pretreated with 100 mM Gallium (III) nitrate. PBS and 100 mM Tris-HCl (pH 4.0) were used as equilibration, binding and washing buffer for the CM10 array and the IMAC-Ga array, respectively. After equilibration, diluted total cell lysates with the buffer were added to each spot and then incubated at room temperature followed by washes with the buffer and water. Arrays were allowed to air dry and a saturated solution of sinapinic acid in 50% (v/v) acetonitrile and 0.5% (v/v) trifluoroacetic acid was added to the spot. The protein chip arrays were analysed using the SELDI ProteinChip System (Ciphergen Biosystems).

Results and discussion Analysis of CM10 array showed differential expression of proteins at 6.2 kDa, 23.5 kDa, 23.6 kDa and 107 kDa in RBL-CCR1 cells stimulated with CCL3 and/or Ag for 1 min (Fig. 2), and at 6.3 kDa, 7.9 kDa, 13.7 kDa and 14.1 kDa in the cells stimulated with CCL3 and/or Ag for 6 h (Fig. 3). Analysis of IMAC-Ga array data showed differential expression of proteins at 9.3 kDa, 9.9 kDa, 17.2 kDa and

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FIG. 2. Comparison of protein expression in RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 1 min using CM10 array. Total cell lysate derived from RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 1 min was applied into spot of CM10 array and analysed by SELDI ProteinChip System. Circle is a sample of each stimulation and bar is the average.

22.3 kDa in RBL-CCR1 cells stimulated with CCL3 and/or Ag for 1 min (Fig. 4), and at 6.2 kDa, 6.4 kDa, 17.2 kDa and 21.4 kDa in the cells stimulated with CCL3 and/or Ag for 6 h (Fig. 5). In the case of the 110 kDa protein (CM10, 1 min stimulation, Fig. 2C), 14.1 kDa protein (CM10, 6 h stimulation, Fig. 3C), 6.4 kDa protein (IMAC-Ga, 6 h stimulation, Fig. 5B) and 21.4 kDa protein (Imac-Ga, 6 h stimulation, data not shown), the highest expression was observed in CCL3-stimulated cells. FceRI-mediated stimulation appears to inhibit the expression. As shown in Fig. 1B, CCL3 induced chemotaxis of RBL-CCR1 cells and IgE/Ag stimulation inhibited the chemotaxis. The expression of these proteins may involve the induction of CCL3-mediated chemotaxis.


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FIG. 3. Comparison of protein expression in RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 6 h using CM10 array. Total cell lysate derived from RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 6 h was applied into spot of CM10 array and analysed by SELDI ProteinChip System. Circle is a sample of each stimulation and bar is the average.

In the case of the 6.2 kDa and 23.5 kDa proteins (CM10, 1 min stimulation, Figs 2A and 2B), 7.9 kDa protein (CM10, 6 h stimulation, Fig. 3A), 6.2 kDa protein (IMAC-Ga, 6 h stimulation, Fig. 5A) and 17.2 kDa protein (IMAC-Ga, 1 min and 6 h stimulations, Fig. 5C), the highest expression was observed in IgE/Ag-stimulated RBL-CCR1 cells. The expression of the proteins was not increased in CCL3stimulated cells, while lower expression was observed in IgE/Ag and CCL3costimulated cells, compared with that in the IgE/Ag-stimulated cells. The result suggests that CCR1-mediated pathway could inhibit the expression of these proteins induced by the FceRI-mediated pathway. In the case of the 9.3 kDa, 9.9 kDa and 22.3 kDa proteins (IMAC-Ga, 1 min stimulation, Figs 4A–C), IgE/Ag and/or CCL3 stimulation decreased the expression.

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FIG. 4. Comparison of protein expression in RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 1 min using IMAC-Ga array. Total cell lysate derived from RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 1 min was applied into spot of IMAC-Ga array and analysed by SELDI ProteinChip System. Circle is a sample of each stimulation and bar is the average.

As shown in Fig. 1A, RBL-CCR1 cells degranulate in response to IgE/Ag and/or CCL3. The decreased abundance of these proteins after the stimulation might indicate that these proteins are granule components in the cells. In the case of the 13.7 kDa protein (CM10, 6 h stimulation, Fig. 3B), costimulation with IgE/Ag and CCL3 enhanced the expression. In a former study, we found that cross-talk between the FceRI-mediated and the CCR1mediated signalling pathways enhanced phosphorylation of extracellular signal regulated kinase (ERK) and of p38 kinase in RBL-CCR1 cells. ERK and p38 kinase are involved in cytokine and chemokine production in mast cells. The observations suggest that the 13.7 kDa protein is a candidate protein expressed following receptor cross-talk in RBL-CCR1 cells. Current efforts are focused at determining the identity of this protein.


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FIG. 5. Comparison of protein expression in RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 6 h using IMAC-Ga array. Total cell lysate derived from RBL-CCR1 cells stimulated with IgE/Ag and/or CCL3 for 6 h was applied into spot of IMAC-Ga array and analysed by SELDI ProteinChip System. Circle is a sample of each stimulation and bar is the average.

In conclusion, CC chemokines are now known to participate in regulating multiple aspects of mast cell biology. In allergic diseases, coengagement with CCRs and FceRI on mast cells appears to involve optimal activation of the cells and thus profound inflammation. In this study, we now show directly that different protein proteomes are expressed in mast cells stimulated via CCR1 and/or FceRI as determined using protein array technology. In parallel efforts we have also compared the protein profiles in the engaged RBL-CCR1 cells using 2D-PAGE and found that expression of many proteins were enhanced by co-engagement with FceRI and CCR1. The identity of the differentially expressed proteins is currently being determined by MALDI/TOF/MS. The protein information would represent the dynamic changes that occur after the mast cells activation and will hopefully be beneficial in new anti-inflammatory drug design.

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References de Paulis A, Annunziato F, Di Gioia L et al 2001 Expression of the chemokine receptor CCR3 on human mast cells. Int Arch Allergy Immunol 124:146–150 Fung ET, Thulasiraman V, Weinberger SR, Dalmasso EA 2001 Protein biochips for differential profiling. Curr Opin Biotechnol 12:65–69 Humbles AA, Lu B, Friend DS et al 2002 The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness. Proc Natl Acad Sci USA 99:1479–1484 Hutchens TW, Yip TT 1993 New desorption strategies for the mass spectrometric analysis of micromolecules. Rapid Commun Mass Spectrom 7:576–580 Laffargue M, Calvez R, Finan P et al 2002 Phosphoinositide 3-kinase gamma is an essential amplifier of mast cell function. Immunity 16:441– 451 Ma W, Bryce PJ, Humbles AA et al 2002 CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine model of allergic skin inflammation. J Clin Invest 109:621–628 Miyazaki D, Nakamura T, Komatsu N et al 2004 Roles of chemokines in ocular allergy and possible therapeutic strategies. Cornea 23:S48–S54 Nakamura T, Ohbayashi M, Toda M, Hall DA, Horgan CP, Ono SJ 2004 A specific CCR3 chemokine receptor antagonist inhibits both early and late phase allergic inflammation in the conjunctiva. Clin Invest Med 27:209A Ochi H, Hirani WM, Yuan Q, Friend DS, Austen KF, Boyce JA 1999 T helper cell type 2 cytokinemediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro. J Exp Med 190:267–280 Ono SJ, Nakamura T, Miyazaki D, Ohbayashi M, Dawson M, Toda M 2003 Chemokines: roles in leukocyte development, trafficking, and effector function. J Allergy Clin Immunol 111:1185–1199 Richardson RM, Pridgen BC, Haribabu B, Snyderman R 2000 Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2. J Biol Chem 275:9201–9208 Sun B, Rempel HC, Pulliam L 2004 Loss of macrophage-secreted lysozyme in HIV-1-associated dementia detected by SELDI-TOF mass spectrometry. AIDS 18:1009–1012 Taub D, Dastych J, Inamura N et al 1995 Bone marrow-derived murine mast cells migrate, but do not degranulate, in response to chemokines. J Immunol 154:2393–2402 Toda M, Dawson M, Nakamura T et al 2004 Impact of engagement of FceRI and CC chemokine receptor 1 on mast cell activation and motility. J Biol Chem 279:48443–48448 Wiesner A 2004 Detection of tumor markers with proteinchip technology. Cur Pharm Biotech 5:45–67

DISCUSSION Rivera: Your CCR3 antagonist is an ocular model. Have you looked at these mice with regards to either passive systemic or passive cutaneous mast cell responses? Ono: The data are consistent in other systems. I don’t think it is strictly an eye phenomenon and I think that there is potential for the antagonism of CCR3 and perhaps other chemokine receptors such as CCR1 or CCR5 in the inhibition of type 1 hypersensitivity reactions. I also point out that should mast cells be key players in autoimmunity (e.g. in multiple sclerosis and other potentially mast cell dependent processes such as ischaemia reperfusion-dependent pathologies), then these small molecular


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antagonists may also have potential in those clinical settings. It is certainly worth careful preclinical examination, in view of the maturity of the drug development programmes in this field and the in vivo pharmacology that is in process. Metcalfe: The other stimulus for mast cell degranulation in eye models is compound 48/80. If you give the CCR3 antagonist, does it also reduce the response to compound 48/80? Ono: That is a great question. I haven’t used 48/80, although we should go back in the lab and do that! But in all seriousness, our laboratory places a high priority on allergen-driven mast cell activation as well as ex vivo studies using human mast cells (remaining intact within their tissue milieu). Williams: Do you think it might be useful to block both CCR1 and CCR3? There are compounds, which we have studied, that will block both receptors. It sounds as if your model is ideally suited to these antagonists. Ono: I would love to do those experiments with you. The reason we were looking at a CCR3 antagonist was because drug companies want an antagonist that is as specific as possible. The best such compounds are not absolutely specific for CCR3; and they do touch CCR1. There are of course concerns about toxicity: one view is that increased specificity for one receptor may lessen the risk of hepatotoxicity. One other complicating factor—as you know—deals with the species specificity of the available small molecule and altered ligand antagonists. Thus, it is not always possible to test a dual specificity compound in the preclinical setting and recapitulate that in humans. If you are referring to the compound described jointly by yourself and James Pease, it was my understanding that the compound might work in humans but not in rodents. Williams: Our antagonist is more potent against CCR1 than CCR3. On another matter, the mechanism by which eotaxin works as a maturation factor is interesting. Have you demonstrated that you can induce that maturation phase with exogenous eotaxin in vitro? Ono: This is what we are doing. There are problems, however. One is that in many of our studies we have used RBL-2H3 cells, which aren’t proper mast cells. In other experiments we have used ex vivo systems because we cannot purify enough mast cells from mouse conjunctiva to do these sorts of experiments. But we are trying these experiments with these imperfect systems, which requires that we interpret the data with caution. However, we do wish to see whether we can up-regulate the mMCP-5 and mMCP-6 expression that we see is not maximal in the eotaxin 1-deficient mice. Stevens: You showed 2D-protein data of mast cells. The mast cell’s granule proteases are ~30 kDa. Presumably you haven’t determined the N-terminal amino acid sequences of the separated proteins. However, is there a noticeable change in the expression of one or more of the ~30 kDa proteins in your cells? Ono: We are looking at this and indeed we are well on the way to determining the nature of the proteome in our experiments via MALDI-TOF analysis. We hope that

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these data will be completed by autumn 2005 at which time we will release such information widely. I agree with you that the mast cell proteases will likely be within the chemokine-induced proteomes. Stevens: Many of the proteases that are expressed in mast cells are positively charged in order to form an ionic complex with the cell’s negatively charged serglycin proteoglycans. mMCP-5 is one of the most positively charged proteins in the mouse’s body. Thus, do you see increased expression of a positively charged ~30 kDa protein in your 2D experiment? Ono: In an ex vivo situation, we can easily give you conjunctiva from chemokinedeficient and wild-type mice. We have done this comparison by RT-PCR, and we could confirm this with immunohistochemistry. Walls: I want to raise the issue of the alterations in expression of proteases in mast cells that you reported. I think this raises the prospect of treatment with chemokine receptor antagonists altering the function of mast cells as well as their degree of activation. Have you had the chance to look at sites other than the conjunctiva to see if the alteration in mast cell proteases induced may be a general phenomenon in the eotaxin 1-deficient mice? Ono: I should say that although I made guarded comments about the potential effect in vivo of chemokine receptor antagonists, these mice have been used in other allergen challenge situations in other tissues. In the late phase response there is a less clear inhibition of eosinophilia than we see, and there is very little effect on the early-phase response. This is not consistent with what I alluded to, that there might be potential for this in other sites of inflammation. Austen: Mark’s mouse is a chemokine knockout, not a receptor knockout. Your drug I presume is directed to the receptor. You shouldn’t mix the two. Ono: You are right. Austen: The receptor data are much more profound than the ligand data. Ono: I agree with you Frank. I should also say that we can extend this away from the mouse into human. We aren’t ready for clinical trials yet, but we are able to take the experiment we do in mice to human. We can take conjunctiva from patients in the hospital and passively sensitize them and measure histamine release. We can look at degranulation directly in human tissue. In the human eye, anti-eotaxin antibody can pretty much silence mast cell activation in human tissue. Brown: I am interested in the relative contribution of mast cells to this process. These receptors are expressed on other inflammatory cells. In your initial experiments you stated that this process is mast cell dependent, on the basis of your studies in W/W v mice. Are you able to reconstitute those mice with mast cells and restore the susceptibility? Ono: We haven’t done this, but we are proceeding towards that with the aid of people like Rick Stevens and others in the room.


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Brown: I’m interested because I want to know whether those mast cells can go to the eye area. Another experiment would be to look at MIP1a-deficient mast cells, and restore W/W v mouse mast cell populations with these. Ono: I think those experiments are extremely difficult because of strain differences. Brown: What is the background of the MIP1a mice? Ono: I’ll have to check. There was something that we were concerned about in that experiment, but it should be done. I am also interested whether mast cells or progenitors will home to the eye in these mice. Hopefully we will get those answers. Pecht: How well characterized are these mucosal mast cells in terms of their phenotype? Ono: The person who did this was M. Allansmith (who actually preceded me in my position at the Schepens Eye Research Institute at Harvard) and they are relatively well characterized. Interestingly, there are many mast cells not only in the conjunctiva but in the choroid as well—surprisingly near the optic nerve. Stevens: The protease phenotype of a mouse mast cell is not fixed (Ghildyal et al 1993). As I mentioned earlier, these cells express varied combinations of at least 16 neutral proteases. Thus, the concept of two distinct populations of mast cells in the mouse is outdated. Generally, those mast cells in the BALB/c mouse that express mMCP-6 also express mMCP-7. Nevertheless, the safranin+ mast cells that reside in the peritoneal cavity of the BALB/c mouse do not express mMCP-7 in contrast to what occurs in the skin (Stevens et al 1994). Three-week old interleukin (IL)3-developed mouse bone marrow-derived mast cells (mBMMCs) express mMCP-6 and mMCP-7. However, the levels of mMCP-6 and mMCP-7 mRNA in these cells slowly decrease the longer the cells are cultured in IL3-enriched medium. mMCP-7 disappears before mMCP-6 in these cultured cells. Based on these data, I would have predicted that the mMCP-7+ conjunctival mast cells in your model would express mMCP-6. A cell that expresses mMCP-7 but not mMCP-6 has not been identified as far as I am aware. As a cautionary note, the level of a protease transcript in a mast cell often does not correlate with the level of its protein. For example, IL3-developed mBMMCs contain more mMCP-5 mRNA than actin mRNA because these cells are desperately trying to become mature mast cells. Nevertheless, because three-week mBMMCs are still immature, the level of mMCP5 protein in these cells is markedly less than in a peritoneal mast cell. As noted in the Ghildyal et al (1993) study, one can alter the levels of mMCP-2 mRNA in mBMMCs within minutes after the cells are exposed to IL10. However, it takes many days of continuous exposure to IL10 to see an appreciable change in the levels of mMCP-2 protein in these cells. Ono: We would love to characterize at a protein level whether or not conjunctival mast cells express protease A, B and C. This is not a trivial task because the

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reagents are special and limited in quantity (as they are often rabbit polyclonals). There is the same heterogeneity in mast cell phenotype that you talked about in the areas where mast cells have been well phenotyped. It wouldn’t surprise me if the mast cells in the eye were to be peculiar; the eye is peculiar in many aspects of its interaction with the immune system. Koyasu: Can you say something about the relative contribution of CCR1 and CCR3 in the mast cell migration? In your experiments using knockout tissue you only showed the eotaxin knockout genotype. In the knockout there is a very low level of histamine release, but when eotaxin is added back you restore this. What happens to the MIP1a knockout tissue? Did you see any difference in terms of the contribution of those two different chemokine receptors? Ono: It is very hard to compare and say which is more important. I would argue that both are critical—only at different stages in mast cell development and activation. In the case of CCR3, I view this is as a terminal differentiation signal in the conjunctiva that primes the mast cell progenitor once it reaches the eye. In the case of CCR1 or CCR5, these are likely to be bona fide co-stimulatory signals. Koyasu: In the wild-type you showed that if you add MIP1a you increase histamine release, but eotaxin doesn’t do this. What happens if you use the MIP1a knockout tissue? Do you see a lower level of histamine release? Ono: There is a lower baseline level that can be augmented by addition of exogenous MIP-1a just as occurs in the experiments using wild-type conjunctiva. The level of rescue is comparable indicating that there is no long-term nonresponsiveness of the mast cell stemming from MIP1a deficiency. References Ghildyal N, Friend DS, Nicodemus CF, Austen KF, Stevens RL 1993 Reversible expression of mouse mast cell protease 2 mRNA and protein in cultured mast cells exposed to IL-10. J Immunol 151:3206–3214 Stevens RL, Friend DS, McNeil HP, Schiller V, Ghildyal N, Austen KF 1994 Strain-specific and tissue-specific expression of mouse mast cell secretory granule proteases. Proc Natl Acad Sci USA 91:128–132

General discussion II

Ono: Our general discussion will revolve around the last three papers. Dr Kawakami, I really liked the IgE painting on the skin you showed. Was this with highly cytokinergic (HC) or poorly cytokinergic (PC) IgE? Kawakami: We tested both. Ono: Is there any strain specificity in the tripling of mast cell numbers that you saw in the response that one antibody was able to promote? If you do the same experiment with HC and PC in many different strains of mice, what happens? Kawakami: We tried a couple of strains. Only NC/Nga mice worked for this experiment. These are mice that are being used as an atopic dermatitis model. Galli: Do you think it is because there is an abnormality of the barrier function of the epidermis in the NC/Nga mouse? IgE is a large protein to get through the epidermis. Kawakami: That’s a good suggestion but there’s no evidence supporting it yet. Stevens: Steve Galli, how did you generate your Rabgef1-null mice? Was the knockout created by replacing one of the gene’s exons with the neomycin gene? Rabgef1 is expressed in many cell types. Thus, it would be more informative if you could selectively knockout this gene in mast cells. As I previously mentioned, we recently placed the Cre recombinase gene downstream of the translation-initiation site of the mouse Mcp5 gene. Thus, if you have floxed the Rabgef1 gene, we could mate our mice with your mice to selectively knock out the Rabgef1 gene in Mcp5-expressing mast cells such as those that reside in the skin and peritoneal cavity? Galli: Yes. We can derive Rabgef1-/- mast cells from knockout mice with a suitable genetic background and then transfer them into a W/W v mouse, in that way isolating the mast cell phenotype associated with the Rabgef1-/- genotype. When we started this work, our concern was that, for other Ras effectors, knockouts have not always produced a marked phenotype (Dhaka et al 2003). Because we first wanted to determine whether we would see any phenotype at all, we decided to use a classical approach. Given what we now know, if we were to do it again, we’d probably make a conditional knockout. Stevens: The central problem with the adoptive transfer approach in which in vitrodifferentiated wild-type mouse bone marrow-derived mast cells (mBMMCs) are injected into the tail veins of W/W v mice is that some tissues (e.g. spleen) end up with more mast cells than they should whereas other tissues (e.g. skin) end up with not enough mast cells. Another major concern with the adoptive transfer approach 145

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is that it generally takes ~8 weeks to at least partially reconstitute the treated animals. Because mice are not given a single cell or cell clone, one has to be concerned about the impact of contaminating cells in the mBMMC cultures. Metcalfe: There was a brief discussion earlier about elevated mediator levels in some of these genetically manipulated mice. The effects of mediators are different if the mediators are released chronically or acutely. The best example I know of is that if we measure plasma histamine levels in mastocytosis patients sometimes we find extremely high levels. These patients appear relatively normal in terms of vascular stability, for example. But in experimental situations where histamine has been infused intravenously to normal volunteers, long before the histamine levels reported in patients with mastocytosis are reached, these volunteers develop a widened pulse pressure, flushing and severe pounding headaches. But these signs and symptoms are not routinely observed in patients with mastocytosis. What is even more instructive is that if we examine patients with mastocytosis who have differing plasma histamine levels, and do dose–responses to histamine in the skin, the responses are the same in patients with mastocytosis as those responses observed in normal individuals with respect to dose response curves where erythema and induration are the measurements (Keffer et al 1989). But if a patient with mastocytosis has a quick rise in histamine level associated with mast cell activation, even if this patient has a high baseline level of histamine, the patient will experience flushing and hypotension. One explanation is tachyphylaxis to histamine; that is patients learn to tolerate higher levels of histamine, providing the rise is gradual. This means that what we infer about histamine and other mediators and their chronic effects on the basis of what we understand concerning acute effects can be misleading. Williams: Eotaxin is primarily a chemokine that recruits eosinophils. You are studying a multiple allergen challenge model in the mouse where you have multiple phases of eosinophil recruitment and activation. The products of eosinophil activation could be having a conditioning effect on mast cells. Thus, when you are using an eotaxin knockout mouse or a CCR3 antagonist you are compromising eosinophil recruitment. How can you be sure that what you are seeing in terms of symptoms is not a consequence of reduced tissue eosinophil numbers and attenuated conditioning of mast cells? Ono: We modified our animal model sensitization scheme, such that we do not challenge topically. We rest the mouse for several weeks. The traditional model was a multiple challenge model involving topical challenge that took place up until very close to the final challenge, to reduce the numbers of eosinophils in situ, so that we could have a robust response in eosinophilia in the late-phase response. This would lead to minimal signal noise so that we could look at the impact of the CCR3 antagonist. We have baseline or slightly elevated eosinophilia because we rest the eyes prior to challenge.


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Williams: So you think that the late phase could be eosinophil-dependent, but the early phase is independent of eosinophils, even in a multiple challenge system. Ono: There are very few eosinophils present in either the un-inflamed eye or in the early phase response (in sensitized mice). However, to say that these few eosinophils have no role in the early phase probably cannot be justified. One would have to do elegant experiments (not yet performed in this model) to answer your question with precision. Williams: During the lifetime of the mast cell in the tissue exposed to allergen challenge, it has been exposed to multiple phases of inflammation, even if you have left an interval. Perhaps this conditions the mast cell. Ono: That is possible. We tried the best we could in that model to avoid this situation, but I can’t discount it. If you have an alternate protocol we would love to try this. Williams: The alternative way of doing this is to use a CCR3 knockout mouse. The results to date using these animals are quite confusing. What is known about mast cells and CCR3 knockouts, or even IgE levels? The effect you showed with elevated IgE was interesting. Ono: We have just got permission from Craig Gerard to get those mice. We will do those experiments, but we haven’t been able to yet. You have been generous to offer to share these with us (which you obtained from Craig) so I hope we can soon do those experiments in London. Williams: Did Alison Humbles in her study see an elevation of antigen-specific IgE, which is what you see? Ono: I should say that in the eotaxin 1-deficient mice, when local sensitization is in the lung, you don’t see what we saw. What we see was very striking. When Mark Rothenberg sensitized the mice with local challenge in the lung, he did not see elevated IgE levels. We do see this in the eye. We will do the experiments on CCR3-deficient mice. I have a question. Do you think this 10-fold increase in allergen-specific IgE might somehow adversely affect the activation of the mast cell in situ? This is why I was so interested in your model where you can bump it up past this. I have spoken to Don MacGlashan about this. MacGlashan: I suggested that this might change the dose—response curve. Rivera: Santa, you cautioned your comments in some respects by suggesting that this might be an organ-specific model. Is there anything that makes the eyes particularly responsive to eotaxin or MIP1a? Might this lead to the mast cell maturing in a different manner? Ono: There is a tremendous literature suggesting that in many facets the eye is unusual in terms of expression of FAS ligand and co-stimulatory models. This is why I said it wouldn’t surprise me at all if mast cells in the eye were unique in phenotype. Is there anything that we know that would make the eye more dependent on CCR3 or eotaxin? The answer is yes. If we do a sensitizing challenge in the eye

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and look at the profile of chemokines that are produced, it is quite different from what one sees when one challenges in the lung. If you do the same kind of analysis for b chemokine protein or RNA in biopsies in patients with chronic allergies, the chemokine that is head and shoulders over any other chemokine is eotaxin. Rivera: I like the idea of a model where one may be talking about organ-specific functions for mast cells. In collaboration with Sarah Spiegel we published ( Jolly et al 2004) that mast cell production of sphingoside-1-phosphate (S1P) causes transactivation of S1P2 receptors on the cell surface of mast cells, which contributes to degranulation. An interesting observation from the Spiegel lab is that one sees a dramatic increase of S1P in the lung upon challenge. S1P production in atopic dermatitis isn’t as marked. Thus, these factors could be important in an organ-specific manner. Stevens: Returning to your eye data, arguably your most striking finding is the presence of only small amounts of mMCP-5 mRNA in your eotaxin-null mouse. If these data can be confirmed, the findings would change our feelings about the use of this transgenic animal in normal and disease states. Is the loss of mMCP-5 a widespread problem? The ears of a normal BALB/c or C57BL/6 mouse have many mMCP-5-expressing mast cells. Thus, did you carry out an RT-PCR analysis of the ears of your eotaxin-null mice to evaluate mMCP-5 mRNA levels? Ono: We haven’t done this experiment but we could, because we have the mice. Where would you look? Stevens: The ear is the easiest tissue to evaluate. We have a rabbit anti-mMCP-5 antibody that you can use to confirm your RT-PCR data. Bradding: What proportion of mast cells in the human conjunctiva express CCR3? In the lung, we have found it is only about 10% of the cells (Brightling et al 2005). Ono: It certainly isn’t 100%. It is more like 30–40%. Bradding: If there was high CCR3 expression in the eye, this would predict different responses to those in the lung. Ono: This is a good point. However, one must keep in mind the CCR3 status of the mast cell progenitor. Depending upon the dosing regimen, a CCR3 small molecule antagonist might have a profound effect—even in the lung or other tissues where say only 10% of the mast cells are CCR3+ due to effects on mast cell progenitors prior to their homing to the end organ. Also, there is the issue of the relative contributions of CCR3+ versus CCR- mast cells in driving earlyphase inflammation. Walls: Can I broaden the discussion further? The title of our meeting is ‘Mast cells and basophils’. What is the role of the basophil in these processes? Might some of the cells which stain with toluidine blue be basophils in your system, and these may express more CCR3 than the mast cells? Ono: The general question of the role of basophils is something that I will refer to Susan MacDonald and Don MacGlashan. In our system, at the electron microscopy level we don’t see many cells that look like basophils.


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Stevens: You showed that the cells express mMCP-2, mMCP-4, mMCP-6 and mMCP-7 which are four prominent constituents of mature mast cells. These data suggest that the cells being evaluated are mast cells rather than basophils. Galli: Most of the standard approaches for using toluidine blue won’t stain mouse basophils in histological sections. However, at the ultrastructural level it ought to be possible to distinguish the two. Ono: We have actually looked at every single mast cell in the conjunctiva of certain animals. Stevens: The mast cells in the skin are stained by safranin because they contain substantial amounts of heparin. Does safranin stain the cells in your eye model? Ono: Yes. Metcalfe: Human basophils are easy to identify morphologically and by their display of characteristic patterns of surface receptors. These human basophils also produce significant amounts of IL4 whereas human mast cells mast cells do not. However, in terms of mouse studies into IgE-dependent inflammation, we ran across a problem when we were looking at one of the lung models of mouse asthma. That is, when we sorted for early IL4-producing cells in dispersed lung cells, about 5% of the IL4-producing cells were mast cells, but the other 95% were a cell type that was difficult to identify (Luccioli et al 2002). They were IgE receptor positive and Kit negative. Some would take up stains and would look basophilic. But the rest looked very neutrophilic. We concluded these cells were most likely basophils. Galli: Because basophils are so difficult to find in the mouse, for many years some investigators thought that the mouse lacked these cells. With a particular method for doing Alcian-blue staining on smears, one can see small numbers of faintly stained blue granules in the cytoplasm of mouse basophils. In the paper Dean just mentioned, the cells that they described were probably basophils. Like with many investigators, attempts to stain them with conventional methods produced what looks like a neutrophil. However, these cells expressed the high-affinity IgE receptor which, in mice, is only expressed on mast cells and basophils. Mouse basophils typically also have low or undetectable levels of surface expression of Kit, as well as a polylobed or bilobed nucleus. Metcalfe: They lack monocyte markers. Galli: We can send you a protocol for a stain that is better at showing mouse basophils. Kawakami: I have a very general question. Whenever people see increased IgE levels anywhere, they tend to think of increased IgE synthesis, not decreased IgE breakdown or clearance. What is known about the mechanism for IgE clearance? Metzger: There is a literature on this. It was thought that it might be mediated by high affinity receptors. Rivera: We generated some data years ago on IgE turnover. However, it was all antigen mediated; it wasn’t really the IgE itself that was the focus.

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Oettgen: I have three questions. Firstly, did you look at antigen-specific responses in G1 and G2a? Ono: We looked at this isotype difference, and showed that there was no effect of chemokine deficiency in either case. This is very intriguing but at this point we do not understand those data. Oettgen: Secondly, did you see that same alteration in responses when you did your earlier studies with W/W v mice? Ono: Yes, we did. Oettgen: Thirdly, do you see the difference after just the intraperitoneal (IP) phase of the experiment, or is this specific to the conjunctival challenge? Did you look at sera after the IP phase or just after the whole protocol? Ono: Both were performed. It actually takes quite a bit of work to get visible IgE measurement. Oettgen: How many IPs are in your protocol before you start doing the eye? Ono: We have different protocols. In this case there were two. Oettgen: If you look right after those two IPs you should have some specific IgE. Ono: We do, but the levels are typically low and it requires longer sensitization to reach high levels. We use allergen-specific IgE levels as a means of determining whether to proceed to final allergen in a specific experiment/protocol. Oettgen: If you look at the IgEs after those two IPs, are they different in the CCR3 knockouts too? Ono: Yes they are specifically elevated in the chemokine knockouts as discussed in my presentation. We do not know yet whether this is the case with CCR3 knockouts. Metcalfe: When you sensitize in your mouse model, do you do it in the standard way with intraperitoneal injection? Ono: We do it by the footpad in the USA but in the UK this is not allowed. Metcalfe: Can you sensitize the mast cells in the eye by directly administering antigen-specific IgE and then add antigen to create an IgE-specific response only in the target organ? In other words, when you do footpad injections you are generating all kinds of systemic immune responses. This approach would isolate responses more clearly to IgE-mediated inflammation and would avoid confounding immune responses generated by footpad injection. Ono: No. Here we don’t do footpad injection, we do IP. Metcalfe: We have always done IP injection. Have you ever tried to isolate the response in the eye by local sensitization? Ono: No. References Brightling CE, Kaur D, Berger P, Morgan AJ, Wardlaw AJ, Bradding P 2005 Differential expression of CCR3 and CXCR3 by human lung and bone marrow-derived mast cells: implications for tissue mast cell migration. J Leukoc Biol 77:759–766


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Jolly PS, Bektas M, Olivera A et al 2004 Transactivation of sphingosine-1-phosphate receptors by FceRI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 199:959–970 Keffer JM, Bressler R, Wright R, Kaliner MA, Metcalfe DD 1989 Analysis of the wheal and flare reactions that follow the intradermal injection of histamine and morphine in adults with recurrent, unexplained anaphylaxis and systemic mastocytosis. J Allergy Clin Immunol 83:595–601 Luccioli S, Brody DT, Hasan S et al 2002 IgE+ Kit- I-A/I-E- myeloid cells are the initial source of IL-4 following antigen challenge in a mouse model of allergic pulmonary inflammation. J Allergy Clin Immunol 110:117–124

The role of phosphoinositide-3-kinase in mast cell homing to the gastrointestinal tract Shigeo Koyasu*†, Akiko Minowa*†, Yasuo Terauchi†‡, Takashi Kadowaki†‡ and Satoshi Matsuda*† * Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, † Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, and ‡ Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Abstract. Phosphoinositide-3-kinases (PI3Ks) are a family of lipid kinases essential in a variety of physiological reactions. A series of gene-targeted mice lacking different PI3Ks and related molecules has enabled us to understand their in vivo roles, particularly those of class IA members. Studies on knockout mice lacking class IA PI3Ks and knock-in mice expressing mutant forms of enzymes have revealed the importance of this class of PI3Ks in mast cell development in the gastrointestinal tract. Here we studied the role of the p85a regulatory subunit, the most abundantly expressed regulatory subunit of class IA PI3Ks, using p85a knockout mice. Development of mast cells in the gastrointestinal tract but not in the skin was severely impaired in mice lacking the p85a regulatory subunit. Stem cell factor (SCF)-mediated signalling functions including proliferative response and chemotactic activities were both impaired in p85a knockout mast cells, likely due to the mast cell deficiency. Mastocytosis upon Strongyloides venezuelensis infection was also impaired in p85a knockout mice. Reconstitution with Th2-conditioned but not untreated bone marrowderived mast cells (BMMCs) restored anti-bacterial immunity, indicating the importance of Th2 response in addition to the recruitment of mast cells in the control of nematode infection. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 152–165

Mast cells are critical effector cells in type 2 helper T cell (Th2)- and immunoglobulin E (IgE)-mediated immune responses (Galli & Hammel 1994, Kawakami & Galli 2002). Although involvement of mast cells in the pathophysiology of various immune diseases, including asthma and fatal anaphylaxis, has been emphasized, mast cells are also critical as sentinels of innate immunity. Upon bacterial infection, tissue mast cells can directly interact with invading bacteria and rapidly secrete a variety of biological mediators such as histamine, leukotrienes, proteases, prostaglandins and cytokines essential for the optimal induction of subsequent 152

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inflammatory and adaptive immune responses (Feger et al 2002, Marone et al 2002). In addition to bacterial infection, mast cells play important roles in parasitic infections as well. In this case, mast cell effector functions are tightly associated with induction of Th2 and IgE production, conferring significant benefits to the host immune reactions against parasites (Finkelman et al 1997). Phosphoinositide-3-kinases ( PI3Ks) are a family of intracellular lipid kinases involved in receptor-mediated signalling in a variety of cell types, including immune cells (Koyasu 2003, Okkenhaug & Vanhaesebroeck 2003). Among them, class IA heterodimeric PI3Ks consist of a catalytic subunit ( p110a, p110b, p110d ) and a regulatory subunit ( p85a, p85b, p55g ). Association with a regulatory subunit is important for stabilizing catalytic subunits and lack of regulatory subunit results in the degradation of catalytic subunits. The role of PI3Ks has been extensively studied in signalling through the high-affinity IgE receptor ( FceRI ), since mast cells are the main effector cells in type I allergic reaction associated with IgE-dependent mechanisms. Although several studies using pharmacological inhibitors of PI3Ks have shown the involvement of PI3K activity in the FceRI signalling pathways, the role of individual family members has been obscure. To precisely examine the functions of class IA PI3Ks, we have employed knockout mouse technology. We and others generated p85a-/- mice deficient for the p85a gene, the most abundantly and ubiquitously expressed regulatory subunit of class IA PI3Ks (Fruman et al 1999, Suzuki et al 1999, Terauchi et al 1999). Due to alternative splicing, in addition to p85a, p55a and p50a are produced from the same gene. Mice lacking only p85a are viable (Suzuki et al 1999, Terauchi et al 1999), while mice lacking all alternative spliced products are unable to survive after birth (Fruman et al 1999). Several groups have generated mice deficient for p110d as well as mice expressing a dominant-negative type mutant of p110d that are viable (Clayton et al 2002, Jou et al 2002, Okkenhaug et al 2002). Knockout mouse studies have shown that class IA PI3Ks are important for the development of gastrointestinal mast cells (Ali et al 2004, Fukao et al 2002a). Furthermore, analyses of knockout mice deficient in various components of the PI3K-mediated signalling pathway have revealed the importance of this pathway in mast cell differentiation and function (Agosti et al 2004, Gu et al 2001, Kissel et al 2000, Nishida et al 2002). The aim of this study is to examine the role of class IA PI3K in mast cell development in the gastrointestinal tract. Steady-state mast cell differentiation and PI3K Mature mast cells are present in all vascularized tissues, especially at sites of internal–external borders such as the gastrointestinal tract and under the epithelial layer of the skin (Galli & Hammel 1994, Kawakami & Galli 2002). Although mast cells are derived from haematopoietic stem cells in the bone-marrow, they leave the

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+/+ +/– –/–

peritoneum ear dermis back dermis mucosa N.D.

stomach submucosa N.D.

muscularis N.D.

jejunum N.D.

ileum colon

N.D. N.D.

0 50 100 (% number of PI3K+/– mast cells) N.D.: not detected

FIG. 1. Mast cell numbers in different anatomical sites. Tissue mast cells were counted under a microscope and the data were expressed as the mean % ± SD compared to the wild-type mice.

bone-marrow as progenitors and complete their maturation after migration into peripheral tissues. The primary signal required for survival and maintenance of mast cells in peripheral tissues is mediated by binding of stem cell factor (SCF) to its receptor c-Kit. Mice with mutations at the SCF locus (Steel locus, Sl ) or the locus for c-Kit (White spotting locus, W ), are severely deficient in mast cells in all organs and tissues (Galli & Hammel 1994, Galli & Kitamura 1987, Kawakami & Galli 2002). We have found that p85a-/- mice lack gastrointestinal mast cells as summarized in Fig. 1. Dermal mast cells were readily observed in the ear and dorsal skin of p85a-/- mice although the cell density was reduced (maximally about 35% reduction in the ear dermis). While mast cells, as defined for positive staining for toluidine blue, alcian blue/safranin or chloroacetate esterase activity, were readily detected in the gastrointestinal tract of wild-type mice, no mast cells were observed in the stomach, jejunum, ileum and colon of the p85a-/- mice (Fig. 1). The number of mast cells in the peritoneal cavity was also reduced by 70–80%. Mast cell development was also examined in knock-in mice expressing a catalytically inactive form of p110d subunit (p110d D910A/D910A mice) (Ali et al 2004). Interestingly, p110d D910A/D910A mice and p85a-/- mice show similar phenotypes although there are


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some differences in mast cell numbers in various organs. These results collectively indicate that among class IA PI3Ks a heterodimer consisting of p85a and p110d is essential for the normal homeostatic production of gastrointestinal mast cells, but dispensable for dermal mast cell differentiation. As mentioned, the SCF/c-Kit system is essential for mast cell development (Galli & Kitamura 1987). Impairment of the c-Kit–PI3K signalling function seems one of the mechanisms accounting for the unique developmental impairment of mast cells in p85a-/- mice. Bone marrow-derived cultured mast cells (BMMC) from p85a-/- mice grew normally in interleukin (IL)3-supplemented medium and expressed FceRI and c-Kit on their cell surface at levels indistinguishable from those of wild type BMMCs. Although IL3-mediated survival and proliferation were unaffected in p85a-/- BMMCs, SCF-induced proliferation of p85a-/- BMMCs was severely impaired (Fukao et al 2002a, Lu-Kuo et al 2000), indicating that the class IA PI3K signalling pathway is important for SCF- but not IL3-induced proliferation of mast cells. Consistent with these observations, knock-in mice with a mutation at the binding site for the p85a subunit of PI3K in the c-Kit gene showed a severe reduction in the number of peritoneal mast cells while dermal mast cells develop normally (Agosti et al 2004, Kissel et al 2000). Although no information is currently available for the development of gastrointestinal mast cells in the above c-Kit knock-in mice, results from these mice are consistent with the observations in p85a-deficient mice and p110d D910A/D910A mice, supporting the hypothesis that defective c-Kit–PI3K-mediated signalling causes the site-selective loss of mast cells in p85a-deficient mice. Selective impairment in the differentiation of gastric and peritoneal mast cells has also been reported in mice deficient for Grb2-associated binder 2 (Gab2), an adapter molecule for various signalling pathways including PI3K (Gu et al 2001, Nishida et al 2002). In Gab2-deficient mice, the numbers of mast cells in the peritoneal cavity, glandular stomach and forestomach are dramatically reduced whereas skin mast cell numbers are unaffected. Defective c-Kit signalling is also observed in Gab2-deficient mast cells, pointing out the involvement of Gab2-mediated signalling downstream of the SCF/c-Kit system (Nishida et al 2002). In contrast to SCF/c-Kit signalling, IL3 signalling was unaffected in Gab2-deficient mast cells as p85a-/- BMMCs (Fukao et al 2002a, Gu et al 2001, Nishida et al 2002). Since Gab2 is a critical positive regulator of PI3K, this site-selective loss of mast cells in Gab2deficient mice is also consistent with the selective loss of gastrointestinal mast cells in p85a-deficient mice and strongly supports the above hypothesis. To examine whether defective production of gastrointestinal and peritoneal mast cells in p85a-/- mice was due to cell-autonomous impairment, we transplanted wild-type bone marrow and BMMCs into p85a-/- mice. Bone-marrow transplantation (BMT) completely restored mast cells in the peritoneal cavity and gastrointestinal tract of p85a-/- mice. Complete restoration of mast cells in p85a-/- mice

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by BMT indicates that the expression of class IA PI3K in bone marrow cells alone is sufficient for the production of mast cells. In contrast, efficiency of reconstitution of gastrointestinal mast cells by BMMC transfer was very low. Partial restoration of gastrointestinal mast cells in p85a-/- mice by BMMC reconstitution points out the possible involvement of environmental defects that preclude the homing of mast cells to the intestine. It is possible that the expression of class IA PI3K in bone marrow-derived cells other than mast cells is critical for homing or survival of mast cells. How do dermal mast cells develop normally in the above mutant mice? Why does the lack of PI3K affect only gastrointestinal mast cells? One possibility is that the SCF/c-Kit system is more critical for the development of gastrointestinal and peritoneal mast cells than for dermal mast cells. IL3 and SCF show overlapping and/or synergistic effects on mast cell differentiation and function (Lantz et al 1998). Several cytokines such as IL4 and IL9 also have critical effects on SCF- and IL3-mediated mast cell differentiation (Godfraind et al 1998, Suzuki et al 2000). Synergistic effects of various cytokines including IL4 on SCF- and IL3-induced proliferation have been observed in p85a-deficient mast cells (Fukao et al 2002a), suggesting that the normal appearance of dermal mast cells is due to the combined effects of various cytokines on mast cell differentiation. Such factors could be absent or present at low levels in the gastrointestinal tract, leading to severe deficiency of mast cells in p85a- and Gab2-deficient mice at the sites where mast cell development is more dependent on SCF. Based on the BMT experiments, these soluble factors may be secreted by bone marrow-derived cells in the intestine. There are other possibilities that may explain the lack of gastrointestinal mast cells in p85a-/- mice (Fig. 2). Lack of chemokine receptors and defects in adhesion molecules on mast cells and surrounding cells may lead to the loss of gastrointestinal mast cells. It has been shown that among integrins, a4b7 integrin is essential for the differentiation and/or homing of mast cells to intestinal mucosa (Artis et al 2000, Gurish et al 2001, Issekutz et al 2001, Pennock & Grencis 2004). Gurish et al (2001) have shown that in b7 knockout mice, the number of mast cell precursors in the small intestine as examined by the limiting dilution method is significantly lower than that of wild-type mice. Consistent with this finding, administration of antibodies against a4b7 integrin or its ligand MAdCAM1 also decreased the number of mast cell precursors in the small intestine. It is, however, unknown whether PI3K activity is involved in the signalling pathway of a4b7 integrin. Much has to be learned regarding the involvement of chemokines in the development of gastrointestinal mast cells. Mast cells are known to express several chemokine receptors including CCRs and CXCRs (Inamura et al 2002, Ono et al 2003, Ruschpler et al 2003). Studies using BMMCs clearly showed that SCF-induced chemotaxis as well as haptotaxis with matrix proteins is sensitive to PI3K inhibitors or lack of PI3K activity, suggesting that class IA PI3K activity is involved in the recruitment of mast


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Bone marrow

Integrins

a4

Chemokines

b7

ex.

Survival signals

CXCR2

MAdCAM-1 ex.

MIP-2

SCF, IL3 etc.

Intestinal lamina propria endothelium

FIG. 2. A model of mast cell development in the intestinal tract. Mast cell progenitors emigrated from bone marrow are likely to develop at the intestinal tract with the aid of integrin–ligand interactions, chemokine recruitment and cytokine receptor survival signals.

cells (Ali et al 2004, Tan et al 2003). It remains to be examined how PI3K activity is involved in such processes. Parasite-induced mastocytosis and PI3Ks Another important developmental phase of mast cells in the gastrointestinal tract is the pathogenic immune phase, when they are rapidly induced upon infection, especially by intestinal parasites. The expulsion of many intestinal parasites, such as Trichinella spiralis and Strongyloides venezuelensis, requires the recruitment of large numbers of mast cells, namely mastocytosis, to the mucosal layer of intestine (Finkelman et al 1997, Shelburne & Ryan 2001). During intestinal parasitic infection, a set of Th2 cytokines is essential for the optimal induction of effector mast cells (Finkelman et al 1997). It is known that the number or density of mature mast cells present in the gastrointestinal tract is much lower than that in the dermis in the steady-state phase. Immature mast cells are present in the submucosa and they migrate to the intraepithelial area upon infection during which time they acquire various effector molecules such as mast cell proteases. Mucosal mastocytosis upon intestinal helminth infection is severely impaired in the c-Kit mutant W/W v mice (Finkelman et al 1997, Lantz et al 1998), confirming that the SCF/c-Kit system is required for mast cell induction in the pathogenic immune phase as well as in the normal immune phase. Mastocytosis is dependent on several cytokines, such as IL3, IL4 and IL10, which determine not only the growth, but also the phenotypes of

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mast cells (Finkelman et al 1997, Shelburne & Ryan 2001). Since these cytokines are produced by Th2 cells, an effective Th2 response is critical for the induction of mastocytosis and subsequent parasite expulsion. Defective anti-parasitic immunity in several gene-targeted mice that lack molecules essential for successful Th2 induction supports this (Finkelman et al 1997). The class IA PI3K is also critical for the differentiation of mast cells in the pathogenic immune phase (Fukao et al 2002a). The p85a-deficient mice demonstrated defects in mastocytosis during infection by Strongyloides venezuelensis. It should be noted, however, that low but significant numbers of mast cells are recruited to the mucosal layer of the intestine in p85a-/- mice, suggesting that upon infection, factors that support mast cell survival or recruitment are produced. It was found that IL3 is produced by mesenteric lymphocytes upon infection, but the amount is much lower in p85a-deficient mice than that in wild type mice. Lower level of IL3 production may lead to modest mastocytosis during helminth infection (Fukao et al 2002a). This is consistent with the impairment of mastocytosis upon S. venezuelensis infection in IL3-deficient and IL3 hyporesponsive mice (Lantz et al 1998). It has been shown that Th2 cytokines are important for terminal differentiation of mast cells that express various effector molecules for parasite expulsion including mast cell proteases and chondroitin sulfate. Production of Th2 cytokines by mesenteric lymphocytes in response to the parasites’ antigens was dramatically impaired in p85a-deficient mice (Fukao et al 2002a). In addition to the impaired IL3 production, defective production of other Th2 cytokines likely leads to the delayed clearance of intestinal nematodes in p85a-deficient mice. Indeed, BMMCs treated with IL4 and IL10 but not untreated BMMCs were able to restore immunity against S. venezuelensis (Fukao et al 2002a). These observations further indicate that the induction of nematode reactive Th2 cells in p85a-deficient mice is impaired. It was further demonstrated that hyperproduction of IL12 by dendritic cells in response to microbial stimuli in p85a-deficient mice causes an enhanced Th1 response (Fukao et al 2002b, Fukao & Koyasu 2003). a4b7 integrin is also essential for effective mastocytosis during parasite infection (Artis et al 200, Issekutz et al 2001, Pennock & Grencis 2004). It has recently been shown that c-kit+b7+ mast cell progenitors are rapidly generated within bone marrow upon T. spiralis infection (Pennock & Grencis 2004). It is thus likely that mast cell progenitors derived from bone marrow migrate to the small intestine with the aid of a4b7–MAdCaM1 interaction. Mastocytosis is impaired in b7 knockout mice infected with Nippostrongylus brasiliensis or T. spiralis, suggesting that production of chemokines alone is insufficient to recruit mast cells to the intestinal epithelia (Artis et al 2000, Issekutz et al 2001, Pennock & Grencis 2004).


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Perspectives The class IA PI3Ks clearly play an important role in mast cell differentiation and recruitment in both steady-state and the pathogenic immune phase. However, several issues still remain to be examined in future studies. One important difference between p110dD910A/D910A and p85a-/- mast cells is the FceRI-mediated signalling functions (Ali et al 2004, Fukao et al 2002a, Lu-Kuo et al 2000). Two groups have reported that mast cells obtained from two independently established p85a-/- mice are stimulated normally via FceRI (Fukao et al 2002a, Lu-Kuo et al 2000). On the contrary, p110dD910A/D910A mast cells are impaired in IgE-mediated degranulation and cytokine production (Ali t al 2004). In addition, p110dD910A/D910A mast cells are impaired in IL3-dependent cell growth (Ali et al 2004), which was not observed in mast cells from p85a-/- mice (Fukao et al 2002a, Lu-Kuo et al 2000). The reason for these discrepancies is currently unknown. More questions remain: how PI3K controls the differentiation of mast cells in distinct tissues, and whether redundancy of different classes of PI3K exists in signal transduction of mast cells. Finding these answers may provide us with possible therapeutic strategies targeting PI3K-mediated signalling in mast cells in allergic disorders such as atopic dermatitis, asthma and anaphylaxis. Acknowledgements We thank Drs. T. Yamada, M. Tanabe, T. Takeuchi and J. Hata for their valuable discussions. This work was supported by a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects, Keio Gijuku Academic Development Funds, the Uehara Memorial Foundation and a Grant-in-Aid for Scientific Research (B) (14370116, 16390146) from the Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research on Priority Areas (C) (13226112, 14021110), a National Grant-in-Aid for the Establishment of a High-Tech Research Center in a Private University, a grant for the Promotion of the Advancement of Education and Research in Graduate Schools, and a Scientific Frontier Research Grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References Agosti V, Corbacioglu S, Ehlers I et al 2004 Critical role for Kit-mediated Src kinase but not PI 3-kinase signaling in pro T and pro B cell development. J Exp Med 199:867–878 Ali K, Bilancio A, Thomas M et al 2004 Essential role for the p110d phosphoinositide 3-kinase in the allergic response. Nature 431:1007–1011 Artis D, Humphreys NE, Potten CS et al 2000 b7 integrin-deficient mice: delayed leukocyte recruitment and attenuated protective immunity in the small intestine during enteric helminth infection. Eur J Immunol 30:1656–1664 Clayton E, Bardi G, Bell SE et al 2002 A crucial role for the p110d subunit of phosphatidylinositol 3-kinase in B cell development and activation. J Exp Med 196:753–763 Feger F, Varadaradjalou S, Gao Z, Abraham SN, Arock M 2002 The role of mast cells in host defense and their subversion by bacterial pathogens. Trends Immunol 23:151–158

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Finkelman FD, Shea-Donohue T, Goldhill J et al 1997 Cytokine regulation of host defense against parasitic gastrointestinal nematodes: Lessons from studies with rodent models. Annu Rev Immunol 15:505–533 Fruman DA, Snapper SB, Yballe CM et al 1999 Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85a. Science 283:393–397 Fukao T, Koyasu S 2003 PI3K and negative regulation of TLR signaling. Trends Immunol 24: 358–363 Fukao T, Yamada T, Tanabe M et al 2002a Selective loss of gastrointestinal mast cells and impaired immunity in PI3K-deficient mice. Nat Immunol 3:295–304 Fukao T, Tanabe M, Terauchi Y et al 2002b PI3K-mediated negative feedback regulation of IL12 production in DCs. Nat Immunol 3:875–881 Galli SJ, Kitamura Y 1987 Genetically mast-cell-deficient W/Wv and Sl/Sld mice. Their value for the analysis of the roles of mast cells in biologic responses in vivo. Am J Pathol 127:191–198 Galli SJ, Hammel I 1994 Mast cell and basophil development. Curr Opin Hematol 1:33–39 Godfraind C, Louahed J, Faulkner H et al 1998 Intraepithelial infiltration by mast cells with both connective tissue-type and mucosal-type characteristics in gut, trachea, and kidneys of IL-9 transgenic mice. J Immunol 160:3989–3996 Gu H, Saito K, Klaman LD et al 2001 Essential role for Gab2 in the allergic response. Nature 412:186–190 Gurish MF, Tao H, Abonia JP et al 2001 Intestinal mast cell progenitors require CD49db7 (a4b7 integrin) for tissue-specific homing. J Exp Med 194:1243–1252 Inamura H, Kurosawa M, Okano A, Kayaba H, Majima M 2002 Expression of the interleukin8 receptors CXCR1 and CXCR2 on cord-blood-derived cultured human mast cells. Int Arch Allergy Immunol 128:142–150 Issekutz TB, Palecanda A, Kadela-Stolarz U, Marshall JS 2001 Blockade of either a-4 or b-7 integrins selectively inhibits intestinal mast cell hyperplasia and worm expulsion in response to Nippostrongylus brasiliensis infection. Eur J Immunol 31:860–868 Jou ST, Carpino N, Takahashi Y et al 2002 Essential, nonredundant role for the phosphoinositide 3-kinase p110d in signaling by the B-cell receptor complex. Mol Cell Biol 22:8580–8591 Kawakami T, Galli SJ 2002 Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2:773–786 Kissel H, Timokhina I, Hardy MP et al 2000 Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 19:1312–1326 Koyasu S 2003 The role of PI3K in immune cells. Nat Immunol 4:313–319 Lantz CS, Boesiger J, Song CH et al 1998 Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 392:90–93 Lu-Kuo JM, Fruman DA, Joyal DM, Cantley LC, Katz HR 2000 Impaired kit- but not FceRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85a gene products. J Biol Chem 275:6022–6029 Marone G, Galli SJ, Kitamura Y 2002 Probing the roles of mast cells and basophils in natural and acquired immunity, physiology and disease. Trends Immunol 23:425–427 Nishida K, Wang L, Morii E et al 2002 Requirement of Gab2 for mast cell development and KitL/c-Kit signaling. Blood 99:1866–1869 Okkenhaug K, Bilancio A, Farjot G et al 2002 Impaired B and T cell antigen receptor signaling in p110d PI 3-kinase mutant mice. Science 297:1031–1034 Okkenhaug K, Vanhaesebroeck B 2003 PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol 3:317–330 Ono SJ, Nakamura T, Miyazaki D, Ohbayashi M, Dawson M, Toda M 2003 Chemokines: roles in leukocyte development, trafficking, and effector function. J Allergy Clin Immunol 111: 1185–1199


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Pennock JL, Grencis RK 2004 In vivo exit of c-kit+/CD49dhi/b7+ mucosal mast cell precursors from the bone marrow following infection with the intestinal nematode Trichinella spiralis. Blood 103:2655–2660 Ruschpler P, Lorenz P, Eichler W et al 2003 High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis. Arthritis Res Ther 5:R241–252 Shelburne CP, Ryan JJ 2001 The role of Th2 cytokines in mast cell homeostasis. Immunol Rev 179:82–93 Suzuki H, Terauchi Y, Fujiwara M et al 1999 Xid-like immunodeficiency in mice with disruption of the p85a subunit of phosphoinositide 3-kinase. Science 283:390–392 Suzuki K, Nakajima H, Watanabe N, Kagami S, Suto A, Saito Y, Saito T, Iwamoto I 2000 Role of common cytokine receptor g chain (gc)- and Jak3-dependent signaling in the proliferation and survival of murine mast cells. Blood 96:2172–2180 Tan BL, Yazicioglu MN, Ingram D et al 2003 Genetic evidence for convergence of c-Kit- and a4 integrin-mediated signals on class IA PI-3kinase and the Rac pathway in regulating integrin-directed migration in mast cells. Blood 101:4725– 4732 Terauchi Y, Tsuji Y, Satoh S et al 1999 Increased insulin sensitivity and hypoglycaemia in mice lacking the p85a subunit of phosphoinositide 3-kinase. Nat Genet 21:230–235

DISCUSSION Austen: In the knockouts, what was the status of b and d ? Koyasu: It depends on which cell type we are talking about. In mast cells, p110d seems to be the major component, whereas in dendritic cells p110b is the major component. In lymphocytes p110d is the major component. In the knockouts, in both cases the levels of p110b and p110d are reduced, although they are not reduced to zero. Austen: When you did your reconstitutions you got a reconstitution of the small intestine, but not the gastric mucosa. Was that a result of the distribution of the worm that you used, or do you have some other explanation? Koyasu: Our interpretation is that it depends on the induction of inflammation. When the mice were infected with helminth, inflammation was induced in the intestine. When we infected with Helicobacter pylori, we observed the migration of the mast cells into the stomach but not to the intestine. Rivera: Have you had the opportunity to measure the amount of activity that is lost? Koyasu: It wasn’t completely lost. In our hands, at least 5–10% of the activity is still there. Rivera: The phenotype of your mice is similar to that of the Grb2-associated binder 2 (Gab2) knockouts. Mast cells from Gab2 knockouts show that 80% of the phosphatidylinositol 3-kinase (PI3K) activity is gone, which mirrors your result quite well. Would your thinking be that the PI3K is primarily associated with this Gab2 complex rather than with other molecules like LAT? Koyasu: We like the idea of an association between Gab2 and PI3K. We haven’t really checked how much activity is associated with one or the other.

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Metcalfe: The knockout may have other systemic effects. Thus, when you characterized mast cells you searched for receptor sets such as a4 and b7 and found that they were not affected, but for each of these ligands did you look at the other side? Was MadCAM affected? Are you missing something in another cell type? Koyasu: This is on our list of things to do. We haven’t looked at integrin-mediated signalling, either. We recently obtained the MadCAM Fc fusion protein, and we are preparing experiments to look at whether adhesion function is affected. Metcalfe: MCP2 production is one of the things that might affect trafficking. Koyasu: After helminth infection the bone marrow-derived mast cells (BMMCs) can go to the intestine. So I am not so worried about the lack of MadCAM. Metcalfe: Do you have baseline data on other adhesion molecules? Cockcroft: In your knockout mice you said you could still get degranulation, in contrast to results from others. You could argue that your cells have a residual activity of 10%, so what would happen if you now pretreat these cells with wortmannin? Koyasu: Possibly, we can block it. But this is not going to show much. If we inhibit the class III PI3K activity, this blocks vesicular trafficking. Cockcroft: In terms of measuring Akt activity, you were down by 90%, suggesting that these cells are not making much phosphatidylinositol-3,4,5-trisphosphate (PIP3). Koyasu: The class 2 and 3 PI3Ks are mostly effective within the cytosolic fraction but not in the plasma membrane. It is not known whether Akt is actually triggered within the cytosol. If we are talking about the Akt activation induced by receptors, class IA PI3K is most likely involved. However, there is always a basal level of PI3K activity in these cells and we still don’t know what controls the activity of class II and III PI3K within the cell. Cockcroft: There is no a priori reason why the class II and III are involved in exocytosis. Koyasu: It is known that thapsigargin-induced degranulation can be blocked by wortmannin (Huber et al 2000). I do not know which PI3K is involved in this process, though. Cockroft: I’m not aware of any hard data showing that class II and III are involved in exocytosis. Class III only makes PI3P, which is involved in endocytic recycling, but not necessarily the degranulation response. This is a different phenomenon altogether. Koyasu: If the PI3K is knocked out it is lethal in yeast. It must be hard to get solid data on this. Rivera: One of the interesting aspects here is that if you are not getting Akt activity, whey isn’t mast cell survival affected? I wouldn’t have predicted that you would have seen normal numbers of mast cells in the bone marrow. Is there any


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evidence that the p110d might be associating with the p55a, rather than p85a? This could be a compensatory mechanism. Koyasu: I showed the receptor-mediated activation of Akt. The basal level is always visible, even in the absence of stimuli. If the signal is operative via FceRI, this can lead to IL3 production, which can support the survival at a later time point. Lee: I wanted to switch the focus back to the lesion in the intestine itself. Wildtype bone marrow replaces mast cells but BMMCs cannot, except if there is inflammation, in which case BMMCs are capable of contributing to intestinal mast cells. A number of other cellular lineages could affect trafficking. Lymphocytes would seem an attractive candidate. Is the effect directly on the mast cell or is there a defect in a critical helper function from another lineage such as intra epithelial lymphocytes (IELs)? Koyasu: That is possible: other types of cells within the intestinal environment might affect a lot of different things. One thing we did was to transfer the bone marrow cells from knockout mice into wild-type and vice versa. When we transferred the bone marrow cells from wild-type mice into knockout mice, we got the normal numbers of intestinal mast cells. As you said, other cells such as IELs may support the survival. This is why we differentially analyse the donor versus recipient mast cells in the same animal. It seems that if we have both wild-type and knockout bone marrow cells together, we always get a lower frequency of mast cells derived from knockout bone marrow even if we have wild-type bone marrow cells in the mice. The number doesn’t change for the precursors from wild-type bone marrow. This argues against a large contribution from other types of cells. Koffer: It is interesting that the knockout cells weren’t able to establish polarity in your chemotaxis assay, but they were able to infiltrate. There must be some additional signals, which they get in vivo. Koyasu: I have no data at the moment. Brown: When you did your original characterization of the mast cells in these mice, in your transcript analysis, did you do a kinetic analysis of the presence of those cells? Stat5-deficient mice have mast cells very early on, but with time those populations begin to disappear. Are those cells in the GI tract at some point and then disappear? It might be that they can go there but this is an issue of long-term survival rather than trafficking. Koyasu: You could be right. When we transfer these cells they go to the intestinal tract. They don’t seem to stay very long. If we really want to expel the worm, we have to keep giving the BMMCs. MacDonald: Why do you see no mast cells in the intestine and stomach, yet you see them in the dermis? Koyasu: That’s a good question. One possibility is that dermal mast cells may have a different cycle of the developmental phase, as Dr Kitamura mentioned. We would like to use fetal liver cells reconstituted in W/W v mice to see whether we can see

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the differences between the two lineages. Another possibility is that the dermis might possess other factors that can support the survival of mast cells. Stevens: Dr Kitamura reported in his presentation that SgIGSF is needed for optimal adhesion of IL3-developed mBMMCs to fibroblasts. You focused your studies on integrins. Have you ruled out the possibility that PI3K is coupled to SgIGSF-mediated signal transduction events? Kitamura: Skin mast cells are present in your knockout mouse, but have you examined the phenotype of these mast cells? MITF mutant mice have about 30% of the normal number of mast cells in the skin. But they don’t express mMCP-6 (mouse mast cell protease 6) in the dermis. Also, the concentration of histamine and heparin was much decreased in skin mast cells of mi/mi mice. Stevens: Dr Kitamura, you showed this adhesion model is important in vitro: is it important in the skin? Kitamura: I don’t have any data on this. Stevens: Dr Koyasu, have you checked for the expression of a mast cell granule protease such as the tryptase mMCP-6? Koyasu: I take your point, but we haven’t really examined any of the mMCP-6 expression. According to the pathologist, from the staining they can’t see any difference between knockout and wild-type mast cells within the skin. In terms of functions, when we first examined the passive systemic anaphylaxis, there was no difference. We also examined in vitro degranulation using BMMCs and didn’t see much difference, so we didn’t examine this in more detail. Dubois: Doesn’t the d kinase knock-in mouse have an inflammatory bowel disease (IBD)-like phenotype? Koyasu: Only one line showed the IBD-like phenotype. The other two lines didn’t show the phenotype. Dubois: Did you look for the presence of Peyer’s patches in your p85 knockout intestine? Koyasu: The Peyer’s patches are present but are smaller than in wild-type (T. Doi and S. Koyasu, unpublished results). Dubois: I think you hypothesized that there might be some effect of IgE in your p85 knockouts. Did you actually get IgE after your helminth infections? Koyasu: We haven’t checked specific IgEs in helminth infections. Kawakami: We had a chance to look at the effect of p110d knockout. We saw the same results as Okkenhaug et al (2004) did. Ono: So it is not an issue of kinase-dead knock-ins versus knockouts. It is something else. Rivera: You are saying that T cells are normal, but are they functionally normal? Koyasu: At least by CD4, CD8 and other markers, there are no differences between the wild-type and p85 knockout T cells. If the cells are stimulated by CD3e they respond as well as wild-type cells.


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Dubois: This was actually shown in the knock-in experiments. In order to see a difference you had to use a suboptimal T cell stimulation using anti-CD3 in the absence of anti-CD28. Koyasu: If there is a difference, it would be in a narrow window. Rivera: I guess this comes down to whether or not the catalytic subunits can react with other regulatory subunits. Koyasu: I believe that the residual activity is mediated by p50a and p110 heterodimer. References Huber M, Hughes MR, Krystal G 2000 Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase. J Immunol 165:124–133 Okkenhaug K, Bilancio A, Emery JL, Vanhaesebroeck B 2004 Phosphoinositide 3-kinase in T cell activation and survival. Biochem Soc Trans 32:332–335

The mast cell and the cysteinyl leukotrienes K. Frank Austen Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA

Abstract. The mast cell has been a fundamental focus for nearly half a century in the effort to understand the biology of the cysteinyl leukotrienes (cysLTs). My initial interest in the cysLTs, once termed the slow reacting substance of anaphylaxis (SRS-A), was based on the findings of others that this activity was elaborated by lung tissue and constricted bronchial smooth muscle in the presence of an antihistamine. We now know that leukotriene C4 (LTC4) is formed when arachidonic acid is cleaved from membrane phospholipids, and metabolized to an epoxide intermediate, LTA4 that in turn is conjugated to reduced glutathione by an integral membrane protein, LTC4 synthase. The LTC4 is exported in an energy-dependent step and subjected to extracellular cleavage of the glutamic acid and then the glycine to provide LTD4 and LTE4, respectively. Mice with targeted disruption of the LTC4S gene are partially protected against plasma leakage elicited in the ear by adaptive immune mast cell activation or in the peritoneal cavity by microbial carbohydrate stimulation of the macrophages. Such mice are also partially protected against pulmonary fibrosis after intratracheal administration of bleomycin. A strain with targeted disruption of the CysLT1 receptor gene is protected against the pathobiological insults that augment microvascular permeability, whereas a strain with targeted disruption of the CysLT2 receptor gene is protected against pulmonary fibrosis. Thus, the expression of these receptors on endothelium, smooth muscle and cells of the haematopoietic lineage such as mast cells, macrophages, and granulocytes extends the possible role of this lipid mediator pathway to both acute and chronic inflammation. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 166–178

In this discussion of the relationship of the mast cell and the cysteinyl leukotrienes (cysLTs), I will first review the events that led to the discovery of the mast cell as the source of the activity originally known as slow reacting substance of anaphylaxis (SRS-A) and now termed the cysLTs. The focus will then turn to the generation, cytokine regulation, and receptor-mediated action of the cysLTs as understood from in vitro studies with cord blood-derived human mast cells and in vivo studies of mice with targeted gene disruptions of biosynthesis or receptor-mediated action. The highlights are the recognition that the cysLTs can mediate chronic as well as 166

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acute inflammation and that they induce cytokines/chemokines in mast cells without exocytosis in a receptor-specific fashion. The mast cell as a source of slow reacting substance of anaphylaxis (SRS-A/cysteinyl leukotrienes) and other eicosanoids The appreciation that the arachidonic acid metabolites now designated as the cysLTs could be provided by activation of mast cells is recognized in retrospect by the early findings that SRS-A and histamine were released together from actively sensitized, perfused guinea pig heart-lung preparations challenged with specific protein antigen (Brocklehurst 1960). The finding that guinea pig lung fragments passively sensitized with plasma fractions enriched for IgG1 but not IgG2 released both SRS-A and histamine separated the response from activation of the complement system and focused directly on tissue mast cells as the source of the activity (Stechschulte et al 1967). The in vivo release of SRS-A into the plasma of actively sensitized guinea pigs protected by prophylactic administration of an H1 antihistamine and challenged with intravenous antigen was recognized by partial purification of SRS-A from plasma and differential bioassay on smooth muscle preparations (Stechschulte et al 1973). A more definitive link of the mast cell to SRS-A generation was provided by the recognition that a novel, heat-labile class of immunoglobulin, IgE, preferentially sensitized mast cells with a longer latent period and duration than the heat stable immunoglobulin species. When rat IgE, identified by its physicochemical and antigenic characteristics, was administered intraperitoneally (i.p.) to rats that were then challenged i.p. with antigen, the reaction generated SRS-A and histamine. Importantly, when the rat peritoneal cavities were pretreated with distilled water or a rabbit antiserum developed against isolated rat peritoneal mast cells to eliminate mast cells, and the rats were then sensitized i.p. with IgE and challenged with hapten-specific antigen, no SRS-A or histamine was generated (Orange et al 1970). The final studies linking the mast cell to SRS-A involved sensitization of monkey lung fragments with atopic serum and activation of mast cells either with ragweed antigen or with anti-IgE. In the reverse-type activations, F(ab’)2 fragments were as active as the intact anti-IgE, whereas the Fab’ fragments were inactive; this discovery implicated bridging of two mast cell-bound IgE molecules in mast cell activation for SRS-A generation (Ishizaka et al 1970). The progression of our physicochemical knowledge of SRS-A, coupled with differential bioassays, allowed us to identify this product in extracts of IgE sensitized and antigen challenged fragments of human lung tissue before it was released. Furthermore, studies in extracts of lung tissue before and after activation showed that SRS-A was not a preformed mediator and that its intracellular accumulation followed by its release continued

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beyond the release of histamine, implying that it was a newly generated product with an export pathway (Lewis et al 1974). The energy-dependent, carrier-mediated export of the cysLT, leukotriene C4 (LTC4), from eosinophils was established 15 years later with the knowledge provided from the structural definition of SRS-A (Murphy et al 1979). The human eosinophil could conjugate LTA4 to reduced glutathione at 4 °C to form intracellular LTC4, which was not released until the temperature was raised (Lam et al 1989). The knowledge that LTC4 was the only intracellular biosynthetic product and that the other components of SRS-A were metabolites (Lam et al 1989, Lewis et al 1980) allowed us to develop a sensitive, competitive, fluorescence-linked immunoassay for the expression cloning of LTC4 synthase (LTC4S) (Lam et al 1994). The only mast cell population that we determined to be devoid of SRS-Agenerating activity when activated immunologically or with calcium ionophore was the rat serosal mast cell. In contrast, these cells were found to be a rich source of a prostanoid, prostaglandin D2 (PGD2) (Roberts et al 1979). PGD2 was then recognized as a major product of IgE-mediated activation of human lung mast cells (Lewis et al 1982). As noted below, the genes for haematopoietic prostaglandin D synthase (H-PGDS) and LTC4S are differently regulated in human and mouse culture-derived mast cells (Hsieh et al 2001, Murakami et al 1995). These key mast cell products, as well as a third, the dihydroxy leukotriene, LTB4 (Lewis et al 1981, Mencia-Huerta et al 1983), are derived from released arachidonic acid, and each acts through at least two separate receptors to mediate the host response to mast cell activation. Biosynthesis of cysLTs and characterization of the CysLT1 and CysLT2 receptors The signal transduction-initiated, calcium ion-dependent translocation of cytosolic phospholipase A2 in concert with serine phosphorylation by the mitogen-activated protein kinase pathway results in release of arachidonic acid from the outer nuclear membrane and the endoplasmic reticulum (Dennis 1994). 5-lipoxygenase (5-LO), which also translocates to the outer nuclear membrane, acts in the presence of an integral membrane protein, the 5-lipoxygenase activating protein (FLAP), to convert the released arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5HPETE) and then to leukotriene A4 (LTA4) (Woods et al 1993, 1995). LTA4 either is hydrolysed by cytosolic LTA4 hydrolase to LTB4 (Funk et al 1987) or is conjugated with glutathione by an integral membrane protein, LTC4S, to form LTC4, the parent of all cysLTs (Lam et al 1994, Yoshimoto et al 1988). After carrier-mediated export of LTC4, cleavage removal of glutamic acid by g-glutamyl transpeptidase or g-glutamyl-leukotrienase and of glycine by dipeptidase provides the metabolites LTD4 and LTE4 (Shi et al 2001).


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Cloning of the human and mouse cDNAs for LTC4S revealed high amino acid identity with FLAP (Lam et al 1994, 1996, Welsch et al 1994). Subsequent cloning of the microsomal glutathione-S-transferases l, 2, and 3, active on xenobiotics as well as LTA4, revealed enough identity to LTC4S to suggest a superfamily of membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) (Jakobsson et al 1999a). The newest member is microsomal PGE2 synthase (M-PGES), which is often induced in concert with prostaglandin endoperoxide synthase-2 (PGHS-2) (Jakobsson et al 1999b). Genomic cloning of both human and mouse LTC4S genes revealed five exons and four introns with an organizational structure identical to that of FLAP. The human LTC4S gene was localized by in situ hybridization to chromosome 5q35, distal to the gene cluster central to the TH2 cell phenotype (Lam et al 1996, Penrose et al 1996). The mouse gene was also found to be located near the TH2 cytokine gene cluster at chromosome 11, a region that is syntenic to human 5q35. Site-directed mutagenesis of LTC4S indicated that two residues, Arg51 and Tyr93, are essential for conjugation of LTA4 to glutathione, with additional residues being involved only in the binding of each substrate. The fact that the mutation of Arg51 to leucine or isoleucine abolished function, whereas the mutation to histidine conserved function suggests that this charged residue is involved in the acid catalysis of LTA4 so as to open the epoxide ring for conjugation. The inactivation with the mutation of Tyr93 to phenylalanine suggests that this residue is required for the base catalysis of glutathione to the thiolate anion for conjugation (Lam et al 1997). The targeted disruption of exons 2 through 4, which includes coding for the catalytic residues, provided a null strain of mice that lack LTC4S activity in peripheral tissues and in bone marrow-derived mast cells (BMMCs), indicating that bifunctional M-GSTs are not an important source of cysLT generation (Kanaoka et al 2001). Two types of human receptors for the cysLTs, designated CysLTl and CysLT2, are 38% homologous in their amino acid sequences and belong to the seventransmembrane G protein-coupled receptor family. They differ in their rank order of receptor-binding affinities as defined in transfected cells, with LTD4 being a log more active than LTC4 for CysLTl yet equally active with LTC4 for CysLT2 (Lynch et al 1999, Heise et al 2000). Their genes map to chromosomes, Xq13-q21 and 13q14, respectively. The fact that both receptors are expressed on airway smooth muscle, microvasculature, and cells of the haematopoietic lineage is compatible with a role for them in acute and chronic host inflammatory responses. The mouse CysLTl and CysLT2 receptors are highly homologous to their human counterparts and exhibit the same distinct ligand binding profile for LTD4 relative to LTC4 in transfected cells; there is weak affinity for LTE4 (Maekawa et al 2001, Martin et al 2001, Ogasawara et al 2002, Hui et al 2001). The mouse genes for the CysLT1 and CysLT2 receptors are localized to chromosomes, X and 14, respectively. Targeted disruption of the gene for the CysLTl receptor was directed to exon 4, which

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encodes almost all the protein, including that for the alternative transcript (Maekawa et al 2002). Peritoneal macrophages from the CysLT1 null strain lacked an intracellular calcium mobilization response, which was evident in their wild-type littermates with a preference for LTD4 over LTC4. The targeting vector for CysLT2 interrupted essentially all the coding, which resides in exon 6. Northern blot analysis with poly A+ RNAs from lung, spleen, and intestine showed the absence of the mature CysLT2 transcript but expression of that for CysLTl receptor, thereby confirming a selective deletion (Beller et al 2004a). Analysis of acute and chronic inflammatory responses in mouse strains respectively null for cysLT biosynthesis and receptor-mediated responses Mouse strains deficient in LTC4S were generated by the injection of targeted 129/Sv ES cell clones into either BALB/c or C57BL/6 mice, which were then extensively backcrossed. LTC4S-/- BMMCs that were activated by cross-linking of FceRI generated no LTC4 but exhibited exocytosis and generation of LTB4 and PGD2 at levels comparable to those from their +/+ littermates (Kanaoka et al 2001). The inability to convert LTA4 to LTC4 resulted in a marked increase in 6-trans LTB4, the degradation product of LTA4, rather than shunting to other end products in FceR1-activated LTC4S-/- BMMCs. Passive cutaneous anaphylaxis (PCA) was examined by sensitizing the mast cells in one ear with an intradermal injection of monoclonal anti-dinitrophenol (DNP) IgE followed in 20 h by intravenous administration of DNP-human albumin. The difference in thickness, measured with microcalipers, of the sensitized and unsensitized ears at sequential time points reflects extravasation of plasma proteins due to the increment in microvascular permeability induced by mediator release from the locally activated mast cells. There was a significant 50% attenuation of ear swelling from 15–60 min in the LTC4S-/strain with kinetics similar to that of the control littermates. The finding that the cysLTs were responsible for half of the ear swelling was unexpected (Kanaoka et al 2001). The contribution of the cysLTs to PCA had been considered minimal because of the lack of inhibition by the receptor blockers in clinical practice, which in retrospect are selective for CysLTl (Inagaki et al 1985, Miura et al 1992). To study the role of the cysLTs in generating microvascular leakage in a different tissue of the BALB/c strain, we administered the yeast cell wall polysaccharide zymosan intraperitoneally to activate the macrophages and Evans blue dye intravenously to label the plasma proteins. Plasma leakage into the peritoneal cavity was assessed by the absorbance of the lavage fluid at 610 nm. There was a significant 50% attenuation of the vascular leakage from 15–60 min. Analysis by reverse phase high performance chromatography confirmed the absence of cysLTs and the presence of LTB4 in the lavage fluid of the null strain. Thus, the cysLTs mediate an acute


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increase in vascular leakage in both the innate and adaptive host inflammatory responses. The discovery that mouse macrophages are an important source of cysLTs and transcribe the CysLT receptors (Maekawa et al 2001, 2002) prompted an examination of a chronic model of inflammation. Bleomycin-induced pulmonary inflammation and fibrosis was chosen because mouse strains respectively deficient in cytosolic phospholipase A2 (cPLA2) and in 5-LO are protected (Nagase et al 2002, Peters-Golden et al 2002). Twelve days after intratracheal administration of bleomycin, the extent of alveolar septal thickening due to accumulation of macrophages and fibroblasts with laying down of extracellular matrix, including collagen, was much less in the LTC4S-deficient B6 strain. By digital image analysis of the lower lobes, the septal thickening was reduced to one-half that observed in the +/+ littermates (Beller et al 2004b). The cysLTs were absent from the bronchial lavage fluid of the LTC4S-deficient strain but abundant in that of their sufficient littermates, whereas the quantities of LTB4 and PGE2 were similar. Thus, in vivo studies revealed a novel function for cysLTs in chronic inflammation that could reflect the distribution of receptors not only to the vasculature but also to cells of the haematopoietic lineage. The pathobiological functions of the cysLTs recognized by their lack of biosynthesis in the LTC4S-deficient strains were further assessed for receptor specificity in strains with targeted gene disruption of each CysLT receptor. As the embryonic stem cell clones with the targeting vectors for the CysLTl and CysLT2 receptors were each C57BL/6, the data available are in this strain. The CysLTl receptor-deficient mice had a significantly attenuated response in both the IgE-mediated, mast celldependent ear swelling (PCA) and in the zymosan-initiated, macrophage-dependent intraperitoneal vascular leakage that was similar in magnitude to that of mice deficient in LTC4S (Kanaoka et al 2001, Maekawa et al 2002). This finding suggests that the acute effects of the cysLTs on the microvasculature are regulated by the CysLTl receptor. In contrast, the CysLTl receptor-deficient strain was not protected and even had an exaggerated bleomycin-induced injury (Beller et al 2004b). The CysLT2 receptor null strain was significantly protected against bleomycininduced septal thickening similar, in magnitude to that of the LTC4S-deficient strain (Beller et al 2004a). That the CysLT2 receptor-deficient strain had normal zymosaninduced intraperitoneal leakage supported a segregation of the receptor functions with CysLT1-mediating acute and CysLT2 mediating chronic pathobiology. However, important caveats are not yet unravelled. The exaggerated bleomycininduced injury in the CysLTl receptor null strain could imply a negative feedback role for the CysLT1 receptor in chronic inflammation. Surprisingly, the PCA response was equally and more than 50% attenuated in each of the receptor-deficient C57BL/6 strains as compared to controls. This finding raises the possibility that CysLTl/CysLT2 receptor heterodimers reside in the ear while CysLT1 homodimers

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dominate in the peritoneum. Human cord blood endothelial cells express a thousand-fold more CysLT2 than CysLTl by quantitative reverse transcriptionpolymerase chain reaction and have a calcium response to cysLTs that is not inhibited by a CysLTl receptor antagonist (Lotzer et al 2003, Sjostrom et al 2003). These early findings for different receptor expression in different tissues have implications for the preferred ligand and for the cytokine/chemokines induced. Cytokine regulation of LTC4S gene expression and CysLT receptor function of human cord blood-derived mast cells Human mast cells, derived from cord blood mononuclear cells cultured for 7 to 9 weeks in stem cell factor (SCF), interleukin (IL)6 and IL10 and sensitized and activated by IgE cross-linking, generate substantial amounts of PGD2 but minimal amounts of cysLTs. Cells primed for 5 days with IL4 generated 25-fold more cysLTs with only a small increment in exocytosis or PGD2, suggesting a selective effect on the 5-LO/LTC4S pathway. Indeed, IL-4 stimulation increased LTC4S gene expression at the transcript, protein and subcellular function levels without increasing the expression of other pathway proteins such as cPLA2, FLAP or 5-LO. Assessment of other TH2 cytokines with co-mitogenic function for these cells in the presence of SCF revealed that IL3 and IL5 also increased cysLT generation but through translocation of 5-LO to the outer nuclear membrane and without a change in expression of any studied pathway protein (Hsieh et al 2001, Ochi et al 1999). With the combination of IL4 and either IL3 or IL5, the IgE-directed generation of cysLTs increased from minimal to equal to that of PGD2, emphasizing that mast cell number and phenotype can be regulated by TH2 cytokines. The priming of these cord blood-derived mast cells with IL4 also increased the IgE-mediated induction of transcripts and protein for regulatory or pro-inflammatory cytokines such as IL13, macrophage inhibitory protein 1a, and tumour necrosis factor a (TNFa) (Ochi et al 2000). With the knowledge that the CysLT receptors are expressed on cells of the haematopoietic lineage (Figueroa et al 2001), we sought their expression on cord blood-derived mast cells by LTD4- and LTC4-initiated calcium flux. Priming the cells with IL4 enhanced their sensitivity to each agonist but lowered the threshold for LTC4 much more than for LTD4, thereby implying a role for a second receptor (Mellor et al 2001). Cytofluorographic staining for surface and intracellular proteins with permeabilization revealed that IL4 increased surface expression of the CysLT2 receptor but not the CysLT1 receptor (Mellor et al 2003). A CysLTl receptor antagonist completely blocked the calcium flux initiated by either LTD4 or LTC4 in IL4 primed cells, suggesting an action on homodimers of CysLTl and heterodimers of CysLTl/CysLT2. That the culture-derived mast cells also express authentic CysLT2


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receptors was appreciated by the ability of the CysLTl receptor antagonist (MK571) to block cysLT-induced expression of IL5 but not of IL8 (Mellor et al 2003). As the cysLTs induce regulatory and pro-inflammatory genes in these mast cells without exocytosis, it seemed possible that their generation might have an autocrine/paracrine effect on gene induction with FceR1 cross-linking. Indeed, expression of IL5 and of TNFa by FceR1 cross-linking was reduced by a significant one-third in the presence of a CysLTl receptor antagonist (MK 571) or a FLAP inhibitor (MK 886) to prevent biosynthesis of cysLTs (Mellor et al 2002). References Beller TC, Maekawa A, Friend DS, Austen KF, Kanaoka Y 2004a Targeted gene disruption reveals the role of the cysteinyl leukotriene 2 receptor in increased vascular permeability and in bleomycin-induced pulmonary fibrosis in mice. J Biol Chem 279:46129–46134 Beller TC, Friend DS, Maekawa A, Lam BK, Austen KF, Kanaoka Y 2004b Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inflammation and fibrosis. Proc Natl Acad Sci USA 101:3047–3052 Brocklehurst, WE 1960 The release of histamine and formation of slow reacting substance (SRSA) during anaphylactic shock. J Physiol 151:416–435 Dennis EA 1994 Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem 269:13057–13060 Figueroa DJ, Breyer RM, Defoe SK et al 2001 Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am J Respir Crit Care Med 163:226–233 Funk CD, Radmark O, Fu JY et al 1987 Molecular cloning and amino acid sequence of leukotriene A2 hydrolase. Proc Natl Acad Sci USA 84:6677–6681 Heise CE, O’Dowd BF, Figueroa DJ et al 2000 Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 275:30531–30536 Hsieh FH, Lam BK, Penrose JF, Austen KF, Boyce JA 2001 T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C4 synthase expression by interleukin 4. J Exp Med 193:123–133 Hui Y, Yang G, Galczenski H et al 2001 The murine cysteinyl leukotriene 2 (CysLT2) receptor: cDNA and genomic cloning, alternative splicing, and in vitro characterization. J Biol Chem 276:47489– 47495 Inagaki N, Goto S, Nagai H, Koda A 1985 Pharmacological characterization of mouse ear PCA. Int Arch Allergy Appl Immunol 78:113–117 Ishizaka T, Ishizaka K, Orange RP, Austen KF 1970 The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung. J Immunol 104:335–343 Jakobsson P-J, Morgenstern R, Mancini J, Ford-Hutchinson AW, Persson B 1999a Common structural features of MAPEG: a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism. Protein Sci 8: 689–692 Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B 1999b Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci USA 96:7220–7225

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DISCUSSION Razin: Could you tell us more about the ligand–receptor interaction? In the leukotriene is the glutathione the domain that interacts with the receptor? Austen: We showed that leukotriene C4 (LTC4), LTD4 and LTE4 are all active, but there is some preferential targeting of smooth muscle cell responses. In LTD4 the glutamic acid is removed, and in LTE4 the glycine is removed so there is nothing left but the cysteine moiety. I believe the peptide is involved in the specificity and that glutathione plays a major role in recognition by the cysteinyl leukotriene2 (CysLT2) receptor. Razin: In other words, you can inhibit the binding by addition of cysteine. Austen: We haven’t looked at that. I’m pretty sure that cysteine will not block. Rivera: In the CysLT1 knockout there is an increase in CysLT1 responses. Is this mediated through up-regulation of the CysLT2 receptor? Austen: There was more bleomycin injury in the CysLT1 receptor null strain. A simple explanation could be that all the CysLTs could simply go to CysLT2. A more elegant possibility is that CysLT1 has a negative regulatory function. Marshall: In terms of the knockout animals you showed, what is the influence of the CysLT1 and CysLT2 defect on the number or development of mast cells? Austen: They are completely normal. Marshall: In the bleomycin experiments do you see a similar increase in the lung as you would normally? Austen: The mast cells are not a player in bleomycin. Marshall: But you see an increased number of mast cells? Austen: Yes, but they are a connective tissue phenotype and they are not very impressive. Marshall: I wasn’t suggesting they were important in the fibrosis. In the cord blood-derived mast cells system, our own experience is that we see good expression of LTC4 synthase even at baseline. Can you comment on the influence of the IL10 on the LTC4 synthase expression? Austen: We haven’t looked at this specifically. Koyasu: You suggested that in the passive cutaneous anaphylaxis (PCA) model the heterodimer was responsible but in the peritonitis model you suggested it was the homodimer. What is the status in the bleomycin model? Austen: In the bleomycin model, the CysLT2 knockout is protected exactly like the enzyme knockout, and the CysLT1 receptor knockout was a little worse. Koyasu: So in the CysLT1 mouse the outcome was worse. In that case, is there any difference between CysLT1 and CysLT2 in their downstream pathways? Austen: The only data we have are in the human mast cell culture system, where there are some differences. The CysLT2 receptor signals for IL8 while the CysLT1 receptor signals for IL5, for example.


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Razin: Where could you find these two receptors? Could you find them on the nuclear membrane? This could help us understand why you trigger the cytokine genes that ignore everything you can see. Austen: We can block the CysLT1 receptors with antagonists that work at the cell membrane. For CysLT2 there is only one molecule that is a partial agonist. It is very useful to us at least in identifying that the CysLT2 molecule is involved. Razin: This doesn’t answer my question. The antagonist could not penetrate into the cell. There is no binding at the beginning. There is no question that they are expressed on the surface of the cells; I am not questioning this. But this is just initiation and then they penetrate into the cells, and the receptor is on the nuclear membrane. Austen: Some of the receptors exist on the cell at time zero. The surface expression of the CysLT2 receptor increases with IL4 priming. We know we can identify them in the cell. Rivera: You showed very nicely that the LTC4 synthetase is up-regulated in the human mast cells by IL4. Since your baseline culturing of these cells is with IL6, does that cytokine have an action on LTC4 synthetase? Austen: It does not. We use IL6 principally to drive proliferation. Metcalfe: Do you know whether CysLTs or PGD2 affect the proliferation of human mast cells in culture? The reason I ask is because we have recently published a study employing high-resolution dye tracking to follow human mast cell proliferation at various stages in culture (Kulka & Metcalfe 2005). One of the curious observations is that IL4, but not IL5 or interferon g, was able to drive a small amount of proliferation in mature human mast cells. This is the only biological response that we have noted so far that changes dramatically with addition of IL4 to these mature mast human cells. Austen: We have looked at a number of cytokines and chemokines that IL4 alone will up-regulate, along with LTC4 synthase. Metcalfe: Do any of these molecules do what IL4 does to mast cells? Austen: No, none of them changed the transcript at that level. The UDP results are kind of interesting. When we started looking at Ca2+ flux in cultured mast cells, the only information was from transfected cells. LTD4 had 10 times the potency of LTC4 in our transfected cells as well. When we began to look at the human culturederived mast cells, we were surprised to find that LTC4 was as active as LTD4. When we primed the human mast cells with IL4 for Ca2+ flux, there were experiments in which LTC4 was a better ligand than LTD4. We went to the databank and found that the closest collection of receptors in terms of sequence to the CysLT receptors are the pyrimidinergic receptors. We dose-responsed the nucleotides and found that UDP was better than any others. UDP crossdesensitized to LTC4 and UDP was blocked by the Merck antagonist for CysLT1. Clearly, with IL4 priming we had spread the specificity. As many of you know G

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protein-coupled receptors are homodimers, but they do form heterodimers. There are good examples but these have all been in vitro. We assumed that is what we saw with our culture-derived mast cells. We also suspect that this is the reason that the PCA reaction was attenuated in both the CysLT1 and CysLT2 null strains, i.e. heterodimeric receptors in the ear but not the peritoneal cavity. Dubois: In your LTC synthase knockout, did you see complete protection of bronchial hyperresponsiveness? Austen: The inhibition is about 50%. Oettgen: Is there any possibility that the IL4-induced synthase underlies aspirin sensitivity in some patients? Austen: We don’t know. The one additional factor I should mention is that we have duplicated this with mouse bone marrow-derived mast cells. This has allowed Bing Lam to show that the ability of IL4 to up-regulate LTC4 synthase requires STAT6 and is independent of p85. Monticelli: Do you see any of the effects seen with IL4 using IL13? Austen: No. IL13 doesn’t do any of the things that IL4 does. But these mast cells generate a lot of IL13. Rivera: Are there IL13 receptors on human mast cells? Austen: Not that we can show functionally. Walls: I want to return to the issue of the percentage reduction in the responses in your various models of inflammation by targeting the leukotrienes or their receptors. With the permeability or septal thickening models, you are seeing a reduction of 50% in the response. You mentioned in reply to an earlier question a figure of 80%. These are high proportions when one thinks of the number of mediators other than leukotrienes that could be involved. Do you think this is a true reflection of the importance of LTC4, or do you think that the treatment interferes with a synergistic interaction between the various mediators? Austen: We think that in the animal models we are looking at pathways for which we have derived the onset, the nature and the limitations of the injury. Thus interventions work more dramatically than they might in outbred human populations. Reference Kulka M, Metcalfe DD 2005 High resolution tracking of cell division demonstrates differential effects of TH-1 and TH-2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression. Blood 105:592–599

Regulation of gene expression in mast cells: micro-RNA expression and chromatin structural analysis of cytokine genes Silvia Monticelli*†, K. Mark Ansel*, Dong U. Lee*1 and Anjana Rao*2 * Department of Pathology, Harvard Medical School, and CBR Institute for Biomedical Research, Boston, MA 02115, USA, and †Department of Biology and Genetics of Medical Sciences, Universita’ degli Studi di Milano, 20133 Milan, Italy

Abstract. Despite deriving from two different compartments of the immune system (myeloid and lymphoid respectively), Th2 cells and mast cells produce the same panel of cytokines, interleukin (IL)4, IL5 and IL13. We have compared the chromatin structure of the RAD50/ IL13/IL4 locus in Th2 cells and mast cells. Th2 and mast cells display strong overlap in their patterns of DNase I hypersensitivity throughout this locus, except that the first intron of the IL13 gene (MCHS) is DNase I hypersensitive only in mast cells and the conserved non-coding sequence (CNS)-1 in the IL4/IL13 intergenic region is DNase I hypersensitive only in Th2 cells (explaining why cytokine expression is impaired in Th2 cells but not in mast cells of CNS-1-deleted mice). We have also examined the role of micro-RNAs (miRNAs) in the development and activation of mast cells and T cells. miRNAs are 21- to 25-nucleotide small RNAs that regulate gene expression posttranscriptionally by targeting protein-coding mRNAs. Using oligonucleotide arrays to analyse miRNA expression in murine T cells and mast cells, we have identified distinctive cell type-specific patterns of miRNA expression as well as changes related to differentiation and cell activation. We are studying the biological functions of selected miRNAs. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 179–190

The cells of the immune system originate from haematopoietic stem cells (HSCs) in the bone marrow, where many of them also mature. HSCs give rise to both myeloid and lymphoid progenitors. The myeloid progenitor is the precursor of granulocytes, macrophages, dendritic cells and mast cells of the innate immune system. Mast cells, whose blood-borne precursors are not well defined, terminate 1 2

Present address: The Salk Institute, La Jolla, CA 92037, USA This paper was presented at the symposium by Anjana Rao 179

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their differentiation in the body tissues, where they are widely distributed and where they orchestrate allergic responses and play a part in protecting mucosal surfaces against pathogens (Galli et al 2005a). The common lymphoid progenitor gives rise to B and T lymphocytes. B lymphocytes differentiate in the bone marrow and T lymphocytes in the thymus; the stages of B and T cell development are defined by sequential rearrangement and expression of heavy- and light-chain immunoglobulin genes, and T cell receptor (TCR) a and b chains, respectively. Small B and T lymphocytes that have matured in the bone marrow and thymus, respectively, but have not yet encountered their specific antigen are called naïve. In the event of an infection, T lymphocytes that recognize the infectious agent are arrested in the lymphoid tissue, where they proliferate and differentiate further into effector cells such as Th1 and Th2 cells that are capable of combating the infection. Mast cells are widely distributed throughout the body and play essential roles in both innate and adaptive immunity. They produce a variety of immune mediators, are critical effector cells in IgE-dependent immediate-type hypersensitivity reactions, and are also well-established as an important source of several cytokines including interleukin (IL)4 and IL13 (Galli et al 2005a, b). These cytokines are also produced by Th2 cells. The IL4, IL5 and IL13 cytokines mediate immunity against parasites and extracellular pathogens and are also central players in the pathophysiology of asthma, allergy and atopic disease (Glimcher & Murphy 2000). The major factor controlling expression of these cytokines is IL4 itself, which acts in a positive feedback loop to promote differentiation of naïve T cells into Th2 cells that strongly up-regulate transcription of IL4, IL5 and IL13 cytokine genes (Ansel et al 2003, Glimcher & Murphy 2000). Analysis of chromatin structure of the IL4 and IL13 genes in Th2 cells and mast cells A key question in gene regulation is whether cell types of different lineages use overlapping regulatory mechanisms to transcribe the same genes. We have addressed this question for the IL4 and IL13 genes by using mast cells and Th2 cells, which represent two different cellular lineages (myeloid and lymphoid, respectively). We and others have previously shown that Th2 cells and mast cells utilize different subsets of trans-acting factors (transcriptional regulators) to activate IL4 and IL13 gene transcription. (i) The transcription factors GATA3 and Maf are essential for maximal IL4 gene expression by Th2 cells, but are not expressed in mast cells (Glimcher & Murphy 2000). (ii) An intact STAT6 signalling pathway is required for Th2 differentiation (Glimcher & Murphy 2000), but mast cells exhibit STAT6-independent IL4 production: bone marrow-derived mast cell (BMMC) precursors in the bone marrow of STAT6-deficient mice could differentiate into mature BMMCs that express IL4 at levels comparable to those of wild-type cells

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FIG. 1. Schematic diagram of the RAD50/IL13/IL4/KIF3a cytokine locus, showing the IL13 and IL4 genes and parts of the flanking RAD50 and KIF3a genes. The direction of transcription is indicated for each gene. Exons are shown as filled boxes and DNase I hypersensitive sites as vertical arrows. The two mast cell-specific MCHS sites are shown in darker grey and the two Th2-specific sites at CNS-1 in lighter grey.

(Sherman et al 1999). (iii) Although IL4 and IL13 gene transcription are dependent on the nuclear factor of activated T cells (NFAT) transcription factor in both mast cells and T cells, the two cell types use distinct NFAT proteins to activate gene expression (Monticelli et al 2004, Tara et al 1995, Weiss et al 1996). A related question is whether transcription of the shared cytokine genes by Th2 cells and mast cells involves the utilization of different cis-regulatory regions. These two cytokine genes are closely linked in an evolutionarily conserved gene cluster located on mouse chromosome 11 and human chromosome 5 (Frazer et al 1997, Loots et al 2000, Seder & Paul 1994) (see Fig. 1). Adjacent to the IL13 gene is the large RAD50 gene, which encodes an essential and ubiquitously expressed DNA repair enzyme; beyond this is the IL5 cytokine gene. The 3¢ end of the RAD50 gene contains a ‘locus control region’ (LCR) for the IL4 and IL13 genes, which was identified by the analysis of transgenic mice bearing BAC transgenes spanning different regions of the RAD50/IL4/IL13 locus (Lee et al 2003). Multi-species sequence comparisons of a 1 megabase region surrounding the IL5/RAD50/ IL13/IL4 locus revealed 90 conserved non-coding sequences (CNS) with presumed regulatory function (Loots et al 2000). The region surrounding the IL4 and IL13 genes, and the 3¢ end of the neighbouring RAD50 gene which contains the LCR, contains several CNSs whose functions have been intensively studied (Fields et al 2004, Lee & Rao 2004, Lee et al 2003, 2005). The biological importance of many of these regions has been established by examining the consequences of deleting them from the mouse genome (discussed further below). Bioinformatic approaches are useful for tentative identification of CNS regions as conserved regulatory regions, but cannot establish whether a given CNS is actually utilized as a regulatory region by any particular cell type. One relatively simple method of assigning potential regulatory function is by examining the accessibility of a CNS region to restriction enzymes and/or DNase I (Nardone et al 2004). At many DNase I hypersensitive (DHS) sites, binding of trans-acting factors leads to

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local disruption of a regular nucleosomal array; thus DHS sites often correspond to physiologically functional cis-regulatory regions (Ansel et al 2003, Gross & Garrard 1988, Nardone et al 2004). Indeed in the RAD50/IL4/IL13 locus, we and others have shown that a remarkably large fraction of CNS regions correspond to DHS regions (reviewed in Ansel et al 2003, Nardone et al 2004). Based on these considerations, we used DNase I hypersensitivity mapping to examine the utilization of CNS regions in the RAD50/IL13/IL4 locus by Th2 cells and mast cells (Monticelli et al 2005b, Solymar et al 2002). We found considerable overlap, but also distinct differences, in the patterns of DNase I hypersensitivity displayed by these two cell types in the RAD50/IL13/IL4 locus. The majority (15 of 19) cis-regulatory elements located within the RAD50/IL4/IL13 locus are utilized in common by Th2 cells and mast cells. We found only two regions of difference: CNS-1, a highly-conserved intergenic sequence located between the IL13 and IL4 genes, is DHS only in Th2 cells, while mast cell-specific hypersensitive sites (MCHS), a moderately-conserved region in the first intron of the IL13 gene, is DNase I hypersensitive only in mast cells. A striking conclusion is that the presence or absence of DHS sites at a CNS region predicts whether or not that CNS region is functional in a given cell type. (i) Thus the highly-conserved sequence CNS-1 corresponds precisely to two DNase I hypersensitivity sites present in Th2 cells (HSS1 and HSS2) but is not a DHS site in mast cells (Monticelli et al 2005b). Consistent with this finding, deletion of CNS-1 resulted in a significant decrease in IL4, IL5 and IL13 production by T cells, but had no effect on cytokine expression by primary mast cells (Loots et al 2000, Mohrs et al 2001). (ii) In contrast, the CNS-2/ DHS V region located 3¢ of the IL4 gene, and the RAD50-C region at the 3¢ end of the RAD50 gene, are DHS in both cell types, and their deletion results in decreased cytokine expression in both cases, identifying both regions as enhancers (Lee et al 2005, Solymar et al 2002). (iii) Another CNS region at the 3¢ end of the IL4 gene, termed DHS IV, is hypersensitive in naïve, Th1 and Th2 cells as well as mast cells; its deletion increases IL4 expression by all four cell types, suggesting that DHS IV functions as a silencer (Ansel et al 2004). Overall these data confirm that bioinformatic searches for CNS regions, followed by DNase I hypersensitivity mapping, constitute a generally feasible method for identifying functional cis-regulatory regions of genes (Ansel et al 2003, Nardone et al 2004). In summary, we have described and compared the chromatin structure of the RAD50/IL4/IL13 locus in Th2 and in mast cells. By showing that CNS-1 is DHS in Th2 cells but not in mast cells, we have solved the paradox of why deletion of this region impaired IL4 and IL13 expression in Th2 cells but was without effect in mast cells. We have also identified two constitutive MCHS in the first intron of the IL13 gene, whose exact function remains to be elucidated. Acquisition of a cytokine-producing phenotype occurs in two steps: (1) developmental signals confer


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locus opening; and (2) activation signals in differentiated cells promote transcriptional gene activation. We could not demonstrate either an acute or a cell type-specific enhancer function for the MCHS region in conventional reporter assays, but propose that it has a role in mast cell differentiation. The MCHS sites develop very early during mast cell differentiation, but are not observed in a ‘precursor’ cell line which lacks the PU.1 transcription factor and so cannot differentiate into mast cells or express the IL4 or IL13 cytokine genes. Thus the MCHS sites could be important for maintaining an accessible configuration of the IL13 locus so as to achieve immediate high-level transcription of the IL13 gene in response to mast cell stimulation. Deletion of the MCHS region in the mouse genome will be required to demonstrate whether this intron region and its associated trans-acting factors contribute in a mast cell-specific manner to IL13 gene expression, by directing locus remodelling during mast cell differentiation and/or by functioning as a mast cellspecific regulatory element in the chromatin context. Regulation of miRNA expression in the murine haematopoietic system MicroRNAs represent a recently-discovered class of small, non-coding RNAs, found in organisms ranging from nematodes to plants to human. Many individual miRNAs are conserved across widely diverse phyla, indicating their physiological importance. The primary transcript (pri-miRNA) is generally transcribed by RNA polymerase II; it contains a typical stem-loop structure which is processed by a nuclear enzyme complex that includes Drosha and Pasha, and releases a 60–110 nucleotide pre-miRNA hairpin precursor (Denli et al 2004). The pre-miRNA is further processed by Dicer to yield the 19–22 nucleotide mature miRNA product, which is then incorporated into the RNA-induced silencing complex (RISC) (Ambros 2004, Bartel 2004, Meister & Tuschl 2004). RISC-bound miRNAs direct the cleavage and/or translational repression of messenger RNAs, thus providing post-transcriptional control of gene expression. Hundreds of miRNAs have been identified in plants and animals, either through computational searches, RT-PCR-mediated cloning, or both. In mammals, more than 200 miRNAs have been reported, and they are estimated to account for at least 1–2% of expressed human genes (Lewis et al 2005, Lim et al 2003). Like many transcription factors, miRNAs are important determinants of cellular fate specification (Hobert 2004). Furthermore, many miRNA genes are located at fragile sites, minimal loss of heterozygosity regions, minimal regions of amplification, or common breakpoints in human cancers, suggesting that miRNAs might play an important role in the pathogenesis of human cancer (Calin et al 2004a, b). MiRNAs have been implicated in biological processes ranging from cell proliferation and cell death during development to stress resistance, fat metabolism, insulin secretion and haematopoiesis (Ambros 2003), but the functions of most mammalian miRNAs

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remain unknown. To understand the role of miRNAs in mammalian development and differentiation, an important starting point is a systematic compilation of miRNAs expressed in individual cell types, especially those derived by differentiation from a common precursor. Because of the wealth of information available about the transcriptional and cellular networks involved in haematopoietic differentiation, the haematopoietic system is ideal for studying cell lineage specification. Many of the common progenitors of haematopoietic cells can be obtained as primary cells from humans and mice, and expanded and differentiated in vitro. We performed a detailed analysis of miRNA expression in diverse haematopoietic cell types from the mouse, using a high-throughput system that allows analysis of many samples with minimal manipulation of the samples themselves. In designing the arrays, we used an expanded version of the array dataset already developed by Krichevsky et al (2003). The new generation of arrays contains 181 gene-specific oligonucleotide probes, corresponding to human and mouse miRNAs as reported in the miRNA Registry (www.sanger.ac.uk/Software/Rfam/mirna). We used these arrays to analyse microRNA expression in cells from several stages of lymphocyte differentiation (Monticelli et al 2005a). Among the cell types compared that are relevant to this symposium were (i) naïve CD4 T ‘helper’ cells, which have exited the thymus, bear the CD4 co-receptor and a mature T cell receptor, and are fully capable of recognizing and responding to antigen; (ii) Th1 and Th2 T helper subsets, which are derived by differentiation from a common precursor, the naïve CD4 T cell, and are characterized by selective expression of the cytokines IFNg and IL4, respectively; (iii) BMMCs; and (iv) a model haematopoietic progenitor cell line (Pu.1-/- ) derived from mice lacking the Ets-family transcription factor PU.1 (DeKoter et al 1998). This cell line differentiates efficiently into mast cells when rescued with PU.1 under conditions where GATA2 expression is maintained; expression of PU.1 in the absence of GATA2 results in commitment to the macrophage lineage instead (Walsh et al 2002). Data from the arrays was validated by extensive Northern blot analysis of ~50 selected miRNAs. With minor exceptions the results of Northern analysis were fully consistent with the array data for most miRNAs, allowing us to identify cell-type-specific differences in miRNA expression as well as differences between miRNAs expressed by precursor cells and their differentiated progeny (Monticelli et al 2005a). We compared miRNA expression in the Pu.1-/- ‘precursor’ cell line, with miRNA expression in BMMCs. Three very different patterns were observed: (i) some miRNAs were expressed at essentially equivalent (low) levels in both the Pu.1-/precursor cells and the fully differentiated BMMCs; (ii) others were expressed at low levels in the Pu.1-/- precursor cells and at higher levels in fully differentiated BMMCs; and (iii) yet others were most highly expressed in the Pu.1-/- precursor cells and were barely detectable in the differentiated BMMCs (Monticelli et al


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2005a). We also compared the expression of the same miRNAs in naïve T cells and fully-differentiated Th1 and Th2 cells (D5 and D10 clones respectively). Several expression patterns were evident also in this case: (i) some miRNAs were expressed at highest levels in the precursor naïve T cells, and were rapidly down-regulated following activation of naïve T cells under both Th1 and Th2 differentiation conditions; (ii) others were equivalently expressed in precursor naive T cells as well as differentiated Th1 and Th2 cells; (iii) others were high in Th1 cells but low in naïve T cells and Th2 cells; and (iv) yet others were poorly expressed in all these T cell types (Monticelli et al 2005a). The miRNA expression pattern of D5 and D10 T cell clones was comparable to that of differentiated primary Th1 and Th2 cells respectively, validating the use of D5 and D10 cells as models for fully differentiated Th1 and Th2 cells. Overall, our results emphasised the interrelation between transcriptional regulation by transcription factors and post-transcriptional regulation by miRNAs (Hobert 2004). It is likely that both pathways of regulation are integral to successful regulation of haematopoietic cell differentiation. Although the predicted targets of most miRNAs in mammals remain to be identified, targeting of key cytokine, cell-surface receptor or transcription factor mRNAs would represent an efficient means for miRNA participation in cell fate decisions. Deciphering the miRNA expression status of cells under different conditions of development and activation and in different disease states will be useful to identify miRNA targets, and alterations in the pattern of miRNA expression may disclose new pathogenic pathways and new ways to target diseases. Acknowledgements This work was supported by a Novartis Foundation bursary to SM, by a Damon Runyon Cancer Research Fund postdoctoral fellowship to KMA DRG-1682, and by NIH grants and a Sandler Program for Asthma Research grant to AR. DUL is a predoctoral fellow of the Ryan Foundation.

References Ambros V 2003 MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113:673–676 Ambros V 2004 The functions of animal microRNAs. Nature 431:350–355 Ansel KM, Lee DU, Rao A 2003 An epigenetic view of helper T cell differentiation. Nat Immunol 4:616–623 Ansel KM, Greenwald RJ, Agarwal S et al 2004 Deletion of a conserved Il4 silencer impairs T helper type 1-mediated immunity. Nat Immunol 5:1251–1259 Bartel DP 2004 MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297 Calin GA, Liu CG, Sevignani C et al 2004a MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA 101:11755–11760 Calin GA, Sevignani C, Dumitru CD et al 2004b Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 101:2999– 3004

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DeKoter RP, Walsh JC, Singh H 1998 PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors. EMBO J 17:4456–4468 Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ 2004 Processing of primary microRNAs by the Microprocessor complex. Nature 432:231–235 Fields PE, Lee GR, Kim ST, Bartsevich VV, Flavell RA 2004 Th2-specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region. Immunity 21:865–876 Frazer KA, Ueda Y, Zhu Y et al 1997 Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region. Genome Res 7: 495–512 Galli SJ, Nakae S, Tsai M 2005a Mast cells in the development of adaptive immune responses. Nat Immunol 6:135–142 Galli SJ, Kalesnikoff J, Grimbaldeston MA et al 2005b Mast cells as ‘tunable’ effector and immunoregulatory cells: recent advances. Annu Rev Immunol 23:749–786 Glimcher LH, Murphy KM 2000 Lineage commitment in the immune system: the T helper lymphocyte grows up. Genes Dev 14:1693–1711 Gross DS, Garrard WT 1988 Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57: 159–197 Hobert O 2004 Common logic of transcription factor and microRNA action. Trends Biochem Sci 29:462–468 Krichevsky AM, King KS, Donahue CP, Khrapko K, Kosik KS 2003 A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9:1274–1281 Lee DU, Rao A 2004 Molecular analysis of a locus control region in the T helper 2 cytokine gene cluster: a target for STAT6 but not GATA3. Proc Natl Acad Sci USA 101: 16010–16015 Lee GR, Fields PE, Griffin TJ, Flavell RA 2003 Regulation of the Th2 cytokine locus by a locus control region. Immunity 19:145–153 Lee GR, Spilianakis CG, Flavell RA 2005 Hypersensitive site 7 of the TH2 locus control region is essential for expressing TH2 cytokine genes and for long-range intrachromosomal interactions. Nat Immunol 6:42–48 Lewis BP, Burge CB, Bartel DP 2005 Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20 Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP 2003 Vertebrate microRNA genes. Science 299:1540 Loots GG, Locksley RM, Blankespoor CM et al 2000 Identification of a coordinate regulator of interleukin 4, 13 and 5 by cross-species sequence comparison. Science 288:136–140 Meister G, Tuschl T 2004 Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349 Mohrs M, Blankespoor CM, Wang ZE et al 2001 Deletion of a coordinate regulator of type 2 cytokine expression in mice. Nat Immunol 2:842–847 Monticelli S, Solymar DC, Rao A 2004 Role of NFAT proteins in IL13 gene transcription in mast cells. J Biol Chem 279:36210–36218 Monticelli S, Ansel KM, Xiao C et al 2005a MicroRNA profiling of the murine hematopoietic system. Submitted Monticelli S, Lee DU, Nardone J, Bolton D, Rao A 2005b Chromatin-based regulation of cytokine transcription in Th2 and mast cells. Submitted Nardone J, Lee DU, Ansel KM, Rao A 2004 Bioinformatics for the ‘bench biologist’: how to find regulatory regions in genomic DNA. Nat Immunol 5:768–774 Seder RA, Paul WE 1994 Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol 12:635–673 Sherman MA, Secor VH, Lee SK, Lopez RD, Brown M 1999 Stat6-independent production of IL-4 by mast cells. Eur J Immunol 29:1235–1242 Solymar DC, Agarwal S, Bassing CH, Alt FW, Rao A 2002 A 3¢ enhancer in the IL-4 gene regulates cytokine production by Th2 cells and mast cells. Immunity 17:41–50


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DISCUSSION Razin: Could you speculate on the 10 most important targets of your miRNA? Monticelli: It’s very hard. When I first decided to take a look at it I used one of the Bartel (Lewis et al 2003) pool of predictions. It was based on human predicted 3¢ UTRs of all the genes. From some of the microarrays that came up from my analysis, some of the potential targets were stem cell factor (SCF) receptor 1, c-Kit or macrophage colony-stimulating factor (M-CSF), so I got really excited. Then there were several problems. Several of the 3¢ UTRs were not annotated. They just took 2000 base pairs after the end of the protein. This sometimes goes into another gene, and when that happens it is not relevant. Razin: Someone indicated to me that one of the main targets is MITF. Is this true? Rao: It is in the list. It is not clear at this point whether this is meaningful. Razin: So there is some kind of regulation of MITF expression at a very early stage. Rivera: One of the aspects of your paper that was particularly interesting to me is whether or not in the DNase hypersensitivity or in the miRNAs regulatory controls there is any strain specificity, either in the available hypersensitive sites or the kinds of the miRNAs that you are able to detect. Monticelli: For the DNase I hypersensitivity, I see the same patterns in C57/BL6 and Balb/C cells. For the miRNA analyses I only used Balb/C. Stevens: How are miRNAs so specific in vivo when only ~6 nucleotides in each miRNA are essential for its recognition of target mRNAs? A very different situation occurs for small interfering RNAs (siRNAs) which use 19–21 nucleotides in the recognition of their targets. Do secondary and tertiary structures in a transcript influence its recognition by the miRNA? For example, do stem–loop structures influence the binding of the miRNA to a transcript? Finally, can you use computer approaches to identify potential targets? Monticelli: What I know comes from a different system, which is bacteria. Bacteria don’t have the same system, but it has been shown that transcription can be regulated by small RNAs that can change the secondary structure of the mRNA. The binding of the small RNA changes the secondary structure of the mRNA, which allows transcription.

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Razin: The miRNA, from my point of view, should regulate expression before the alternative splice. It should work only on the immature RNA. Monticelli: It binds the 3¢ UTR. Brown: One of the interesting aspects of your data is that resting mast cells have a very open locus that is parallel to the activated mast cell. This goes along with the idea that I think about a lot, which is that mast cells are poised to respond more immediately than T cells. T cells need activation and their kinetics of cytokine expression is much delayed compared with mast cells. I also have a couple of questions. First, you suggested that the regions of hypersensitivity in the 3¢ end of the RAD50 gene were locus control regions (LCRs): do you have any data to support this? Rao: The data on LCR function aren’t our results, they are from Richard Flavell. The empirical definition of an LCR is that it controls expression of a linked gene in a copy number-dependent and integration site-independent fashion. These criteria are certainly fulfilled by the 3¢ end of the RAD50 gene which contains the hypersensitive sites. Brown: He said this about conserved non-coding sequences (CNS-1) also, didn’t he? Rao: No, the CNS-1 data are from Richard Locksley. CNS-1 doesn’t seem to have the same function as the RAD50 LCR. CNS-1 is an individual intergenic enhancer that controls IL4 and IL13 gene expression, while the RAD50 LCR is a big block, some 25 kb long, containing several enhancers. Brown: You said that nuclear factor of activated T cells (NFAT) and signal transducer and activator of transcription (STAT) are bound there. Are these both inducible? Rao: Yes. Brown: Yet at least one of these sites is constitutive. Rao: That’s the peculiarity of open chromatin structures. Bill Paul and Toshi Nakayama have recently published papers (Zhu et al 2004, Yamashita et al 2004) showing that if GATA3 is removed after Th2 cells have fully differentiated, the cells will still produce IL4 and will still show increased histone acetylation, indicating an open chromatin structure. Clearly, there is a whole sequence of steps involved. Inducible factors come in and direct synthesis of the lineage-specific transcription factors, which in turn will be replaced in the longer term with factors that may be completely independent of the cell type but maintain chromatin accessibility, saying that this is a gene that needs to be transcribed. Koyasu: You show that in resting mast cells there is a specific hypersensitive site in the IL13 gene. Does this site actually bind NFAT1? Rao: We don’t know what factors bind to the intronic mast cell-specific hypersensitive sites (MCHS) sites in the IL13 gene in mast cells.


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Galli: This is a theoretical question. In protein overexpression studies, at the high end there can be a problem with the overexpression leading to misleading results, although it can be a very useful approach in general. Is there enough experience now with miRNA overexpression, as opposed to attempts to eliminate them, to know which is the more informative approach? Monticelli: I don’t think there is enough known about this. Many of the papers that have been published to date have this system of overexpressing miRNA and putting in a luciferase reporter gene with a target site engineered into the 3¢ UTR. I liked the Bartel paper (Chen et al 2004) in which he showed that overexpression of miR-181 skewed haematopoietic differentiation of bone marrow precursors towards the B cell lineage. One can also knock out miRNA function by using 2¢O-methyl oligoribonucleotides (Meister et al 2004) and these also have functional effects. Rao: The difficulty with knocking out miRNAs is that they often come as families of several related genes which may or may not reside in the same genomic location. So deleting any individual miRNA may not be sufficient to produce a detectable phenotype. Ono: Did you look at the two mast cell-specific hypersensitive sites that you have mapped during BMMC differentiation? Which comes on first? Monticelli: I wish I could do this experiment. If any one can give me an idea about how I can isolate enough precursors and cells at different stages of differentiation, I’d be glad to know. Galli: I’m guessing that is why you are using embryonic stem (ES) cells, in part. Ono: I have a question about the hypersensitive site in IL13. Surely you must be able to inspect a sequence plus or minus 500 base pairs of the site you mapped, to precisely identify where it is. If you look at that sequence, what do you see? Monticelli: I was able to map it a little more closely with restriction enzyme hypersensitivity. I looked at the sequence and there were putative GATA and NFAT sites. These were common. These hypersensitive sites were constitutive, though, and I wasn’t able to see any enhancer activity. Ono: Have you put it in front of a reporter construct and can you activate it by mast cell differentiation signals? Monticelli: I did that, and didn’t see any enhancer activity in a reporter system. Ono: Do the boundaries that you have mapped correspond in terms of higherorder structure to matrix attachment agents? Rao: I have talked to Terumi Kohwi-Shigematsu who has this matrix binding protein called SATB1 which binds AT-rich sequences. There are clearly AT-rich regions that are in interesting positions to make things loop out. She is in the process of figuring out how to visualize where the loops are.

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References Chen CZ, Li L, Lodish HF, Bartel DP 2004 MicroRNAs modulate hematopoietic lineage differentiation. Science 303:83–86 Zhu J, Min B, Hu-Li J, Watson CJ et al 2004 Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat Immunol 5:1157–1165 Yamashita M, Ukai-Tadenuma M, Miyamoto T et al 2004 Essential role of GATA3 for the maintenance of type 2 helper T (Th2) cytokine production and chromatin remodeling at the Th2 cytokine gene loci. J Biol Chem 279:26983–26990 Meister G, Landthaler M, Dorsett Y, Tuschl T 2004 Sequence-specific inhibition of microRNAand siRNA-induced RNA silencing. RNA 10:544–550

The involvement of Bcl-2 in mast cell apoptosis Cellina Cohen-Saidon and Ehud Razin1 Department of Biochemistry, Hebrew University—Hadassah Medical School, POB 12272, Jerusalem 91120, Israel

Abstract. Apoptosis or programmed cell death plays an important role in a wide variety of physiological processes. Apoptosis is regulated by proteins of the Bcl-2 family consisting of both anti-apoptotic and pro-apoptotic factors. The direct involvement of the Bcl-2 protein family in the process of mast cell apoptosis has not been clarified. We have used a single-chain antibody (scFv) raised against Bcl-2 derived from human phage-display antibody library. The addition of TAT sequence, which is responsible for translocation through the membrane, endows the anti-Bcl-2-scFv with the ability to penetrate living cells. The association of anti-Bcl-2-scFv-TAT with intracellular Bcl-2 leads to neutralization of Bcl-2 and eradication of its anti-apoptotic activity in two types of mast cells and in a human breast cancer cell line. Moreover, we found by mass spectrometry and co-immunoprecipitation assay that heat shock protein 90b (Hsp90b) forms a complex with Bcl-2 in mast cells. Thus, understanding the network of interactions between Bcl-2 and non-Bcl-2 family members might help in development of more specific drugs and cancer therapy. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 191–199

Mast cells and apoptosis Mast cells are long-living cells of myeloid origin that are known as multifunctional effector players of the immune system (Serafin & Austen 1987, Galli 1990). Since their discovery by Paul Ehrlich over 100 years ago (Ehrlich 1877), many scientific techniques have been applied to try to unravel their origin, structure and function. It has taken a great deal of effort to find out where mast cells come from, how long they live in the tissues and how they are perpetuated. Hunt (1957) showed very clearly that mast cells were able to regenerate and proliferate in the rat peritoneum after being degranulated following injections of compound 48/80. They observed the sequential repopulation of peritoneal tissue by young mast cells which become 1

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fully mature in 17 days (Hunt 1957). Moreover, the number of mast cells in tissues has been found to be constant under normal conditions, which probably reflects an equilibrium between cell proliferation, migration and death (Finotto et al 1997, Mekori et al 2001). Mast cells need growth factors for their survival and differentiation, as shown by withdrawal experiments of growth factor from culture (Mekori et al 2001, Razin et al 1984). Such elimination of growth factors leads to cell apoptosis which can be prevented by the addition of the withdrawn growth factors: Kit ligand (also known as stem cell factor [SCF]), interleukin (IL)3 and IL6 (Kambe et al 2001, Yanagida et al 1997, Mekori et al 2001). However, at the molecular level little is known about mast cell apoptosis. These biochemical events leading to programmed cell death in mast cells have recently started to be explored. Bcl-2 and mast cells The Bcl-2 family of proteins are central regulators of apoptotic death consisting of both anti-apoptotic (Bcl-2, BclXL, Mcl-1 and A1/Bfl-1) and pro-apoptotic (BclXS, Bax, Bak and Bok/Mtd) factors (Adams & Cory 2001, Belka & Budach 2002). The ability of many Bcl-2 family members to form homo- as well as heterodimers through their Bcl-2 homology (BH) domains is important for the activation of specific functions such as causing changes in mitochondrial membrane potential and initiating the process of apoptosis, and also for neutralizing these functions in the cells (Danial & Korsmeyer 2004, Cory et al 2003). The involvement of Bcl-2 family proteins in human mast cell survival was suggested by the observation that SCF induced elevation of the anti-apoptotic factors Bcl-2 and BclXL in these cells (Mekori et al 2001). Several studies on mast cell leukaemia show overexpressed levels of Bcl-2, pointing out the importance of this anti-apoptotic protein in mast cell homeostasis (Cervero et al 1999). Other experiments reveal that upon stimulation via the aggregation of highaffinity IgE receptors (FceRI), mouse mast cells synthesize the anti-apoptotic component A1 rather than Bcl-2 (Xiang et al 2001). However, it has not been determined whether A1 plays a direct role in mediating cell survival in these cells. Later it was found that only monomeric IgE binding to FceRI suppresses the apoptosis induced after growth factor deprivation (Asai et al 2001). This induction of mast cell survival was observed without detectable changes in the expression of other members of the Bcl-2 family of proteins (Asai et al 2001). Thus, there is still uncertainty with regard to the involvement of Bcl-2 in promoting mast cell survival. In order to decipher the role of Bcl-2 in mast cell apoptosis, we addressed this by using a human semi-synthetic phage display antibody library (Cohen-Saidon et al 2003). Phage display libraries of peptides have been used since mid-1980 as a highly effective method for selection of interacting partners. The display of antibody fragments as single-chain Fv (scFv) on phage and the subsequent antigen-driven selection has

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provided a new way for the development of human monoclonal antibodies with potential therapeutic applications (de Wildt et al 2000). After selection against recombinant Bcl-2, we isolated specific anti-Bcl-2-scFv and modified them by genetic engineering (Cohen-Saidon et al 2003). To the new single chain was added a signal sequence derived from the TAT protein of HIV, which enables it to penetrate the cell membrane. This novel antibody against Bcl2 was shown to translocate into cells and to bind specifically to Bcl-2. This interaction at a Bcl-2 region including the BH1 domain leads to neutralization of the Bcl-2 anti-apoptotic activity as observed by the decrease in the mitochondrial membrane potential and apoptosis of two types of mast cells (Cohen-Saidon et al 2003). Regulation of Bcl-2 activity The consensus is that binding of the pro-apoptotic factors to Bcl-2 is modulated through several pathways: interaction with Bcl-2 family proteins (Cory et al 2003); post-translational modification, such as phosphorylation on serine and threonine residues (Blagosklonny et al 1996); and, discovered most recently, through interaction with proteins not belonging to the Bcl-2 family (Dias et al 2002, Lin et al 2004). The consensus is that binding of the pro-apoptotic factor Bax to Bcl-2 leads to an increase in the pro-apoptotic ratio in the cell and Bcl-2 is unable to inhibit the programmed cell death (Gross et al 1999). The findings of research into the effect of phosphorylation of Bcl-2 are contradictory. It has been reported that when Bcl-2 is phosphorylated on Ser70 in cells dependent on growth factors, apoptosis was prevented (Poommipanit et al 1999). In contrast, the chemotherapeutic drug paclitaxel leads to the phosphorylation of Ser 70 on Bcl-2, and was shown to neutralize its anti-apoptotic activity and so caused cell apoptosis (Pratesi et al 2001, Blagosklonny & Fojo 1999). Recently it was reported that Bcl-2 is converted from protector to killer by interaction with the nuclear receptor Nur77/TR3 (Lin et al 2004). Using mass spectrometry we found that Hsp90b binds Bcl-2 in mast cells (Cohen-Saidon et al, manuscript in preparation). Previously it was reported that vascular endothelial growth factor (VEGF) promotes the survival of leukaemic cells by Hsp90mediated induction of Bcl-2 expression and apoptosis inhibition (Dias et al 2002). Our results demonstrate that upon dissociation of the complex Bcl-2-Hsp90b by the specific inhibitor of Hsp90, geldanamycin (GA), cytochrome c is released from the mitochondria and caspase 3 is activated through the apoptotic pathway (C. Cohen-Saidon et al, unpublished). Clarification of this pathway may lead to possibilities for future clinical intervention for allergy and mast cell diseases. Indeed, it has been shown that the GA analogue 17-allylamino-17-demethoxygeldanamycin (17-AAG) is effective in down-regulating mutated, constitutively activated KIT protein in human mast cells (Fumo et al 2004).

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References Adams JM, Cory S 2001 Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 26:61–66 Asai K, Kitaura J, Kawakami Y et al 2001 Regulation of mast cell survival by IgE. Immunity 14: 791–800 Belka C, Budach W 2002 Anti-apoptotic Bcl-2 proteins: structure, function and relevance for radiation biology. Int J Radiat Biol 78:643–658 Blagosklonny MV, Fojo T 1999 Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer 83:151–156 Blagosklonny MV, Schulte T, Nguyen P, Trepel J, Neckers LM 1996 Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res 56:1851–1854 Cervero C, Escribano L, San Miguel JF et al 1999 Expression of Bcl-2 by human bone marrow mast cells and its overexpression in mast cell leukemia. Am J Hematol 60:191–195 Cohen-Saidon C, Nechushtan H, Kahlon S et al 2003 A novel strategy using single-chain antibody to show the importance of Bcl-2 in mast cell survival. Blood 102:2506–2512 Cory S, Huang DC, Adams JM 2003 The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22:8590–8607 Danial NN, Korsmeyer SJ 2004 Cell death: critical control points. Cell 116:205–219 de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM 2000 Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol 18:989–994 Dias S, Shmelkov SV, Lam G, Rafii S 2002 VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition. Blood 99:2532–2540 Ehrlich P 1877 Beitrage zur Kenntnis der Anilinfarbungen und ihrer Verwendung in der Mikroskopischen Technik. Arch Mikr Anat 13:263 Finotto S, Mekori YA, Metcalfe DD 1997 Glucocorticoids decrease tissue mast cell number by reducing the production of the c-kit ligand, stem cell factor, by resident cells: in vitro and in vivo evidence in murine systems. J Clin Invest 99:1721–1728 Fumo G, Akin C, Metcalfe DD, Neckers L 2004 17-Allylamino-17-demethoxygeldanamycin (17AAG) is effective in down-regulating mutated, constitutively activated KIT protein in human mast cells. Blood 103:1078–1084 Galli SJ 1990 New insights into ‘the riddle of the mast cells’: microenvironmental regulation of mast cell development and phenotypic heterogeneity. Lab Invest 62:5–33 Gross A, McDonnell JM, Korsmeyer SJ 1999 BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911 Hunt EA HT 1957 Mitotic activity of mast cells. Proc Soc Exp Biol Med. 94:166–169 Kambe M, Kambe N, Oskeritzian CA, Schechter N, Schwartz LB 2001 IL-6 attenuates apoptosis, while neither IL-6 nor IL-10 affect the numbers or protease phenotype of fetal liver-derived human mast cells. Clin Exp Allergy 31:1077–1085 Lin B, Kolluri SK, Lin F et al 2004 Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell 116:527–540 Mekori YA, Gilfillan AM, Akin C, Hartmann K, Metcalfe DD 2001 Human mast cell apoptosis is regulated through Bcl-2 and Bcl-XL. J Clin Immunol 21:171–174 Poommipanit PB, Chen B, Oltvai ZN 1999 Interleukin-3 induces the phosphorylation of a distinct fraction of bcl-2. J Biol Chem 274:1033–1039 Pratesi G, Perego P, Zunino F 2001 Role of Bcl-2 and its post-transcriptional modification in response to antitumor therapy. Biochem Pharmacol 61:381–386 Razin E, Ihle JN, Seldin D et al 1984 Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan. J Immunol 132:1479–1486 Serafin WE, Austen KF 1987 Mediators of immediate hypersensitivity reactions. N Engl J Med 317:30–34


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Xiang Z, Ahmed AA, Moller C et al 2001 Essential role of the prosurvival bcl-2 homologue A1 in mast cell survival after allergic activation. J Exp Med 194:1561–1569 Yanagida M, Fukamachi H, Takei M et al 1997 Effect of a chymotrypsin-like inhibitor, TPCK, on histamine release from cultured human mast cells. J Pharm Pharmacol 49:537–541

DISCUSSION Pecht: I have a question regarding the association that proceeds via the N domain. This is also the domain for ATP and geldanamycin (GA) binding. Could you induce the same phenomenon that GA induces by using non-hydrolysable analogues of ATP? Razin: Good question. That’s on our list of things to do. Pecht: How is the anti-apoptotic activity related to this? Razin: It depends what causes it. If it is through Hsp90, ATPase activity is stopped. Dean Metcalfe, you have used GA on mastocytosis patients. Can you comment on this? Metcalfe: Geldanamycin has been used in clinical trials for various malignancies. It is not entirely clear how it preferentially kills tumour cells. It does preferentially lead to apoptosis of mast cells that bear mutated Kit. If we take mast cells from a person with mastocytosis and culture them in GA, this kills these cells with mutated Kit preferentially (Fumo et al 2004). The data are encouraging and we are in the early phase of a clinical trial; and we hope to soon begin treating patients with mastocytosis with GA. It is a reasonably safe agent. Razin: I would like to emphasize that all our work was performed on mast cells. I don’t know whether this is a general mechanism or not. Koyasu: You know that heat shock protein 90 (Hsp90) is associated with many different types of kinases. If you add GA you can wipe out many kinases. It is complicated to interpret the results of these sorts of experiments. You said that the Hsp90b is specifically associated with Bcl2. How can you exclude Hsp90a? Did you use a specific antibody for the b? Razin: There is no antibody specific for a or b. In order to address this we are analysing what is associated just by mass spectrometry. It is clear that there is no a there. Koyasu: Can you do the in vitro experiment using the purified Hsp90? Razin: We did this with Hsp90 a and there is no association in vitro. Koyasu: They are closely related in terms of sequence. Can you comment on which sequence is involved in the association? Razin: Let me say that they are coded by two different genes, both called Hsp90. The differences aren’t on the N-terminus but in what we call the M-domain. We have a model of how the M-domain associates with Bcl-2. We can’t find this with a because there are differences in the middle domain. Koyasu: Are you suggesting they are present as monomers?

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Razin: It is not a heterodimer and it is not a dimer; it is a monomer. Koyasu: If Hsp90 molecules are taken from the cell they are dimers. Razin: No. I am talking about what happens in vivo. If we immunoprecipitate Hsp90 it is a dimer between a monomeric Hsp90 and a monomeric Bcl-2. Bcl-2 could also form a homodimer. Koyasu: So you are suggesting that the binding of Bcl2 to Hsp90 changes the formation. Razin: When you are talking about association between two proteins, this sort of thing happens a lot. I am working on some other proteins that are associated with MITF. One of them is Hint which you can find as a dimer but when it is associated with MITF it is a monomer. Rivera: Hsp90 seems to act more as a chaperone protein in some cases, and is important in maintaining the conformation of a protein in a functional or nonfunctional state. In other cases it seems to be absolutely critical for activity of that protein. If you put in your GA compound, is the Bcl-2 activity affected in any way? Can this be measured? Razin: Bcl-2 isn’t an enzyme so the way we have to measure its activity is by measuring cytochrome C release from mitochondria. This is the only way we can do this. Pecht: It is residing in the membrane of the mitochondria. It has a transmembrane domain, but you don’t know what really couples the effect that occurs downstream. Razin: We know that in order for the dissociation between Hsp90b and Bcl-2, two sites need to be phosphorylated. It occurs in the M phase of the cell cycle. Pecht: Do you have any insight into the cross-talk between Bcl-2 and the mitochondria? How is the cycle of cytochrome C release triggered? Razin: We just check physiology—what happens to the membrane potential. Pecht: Bcl-2 undergoes homo or heterodimerization. How is phosphorylation of these particular sites related to heterodimerization of Bcl-2? Razin: We don’t know. The other thing that is interesting is that when the ‘smart missile’ is sent into the cell, it is bound to the BH1 domain and still we see association between Hsp90b and Bcl-2. It is complicated. Walls: Could I ask about this ‘smart missile’ antibody: how does it penetrate the mast cell? Is there something specific about Bcl-specific antibodies? Razin: I don’t think so. However, I have seen a report in the literature where researchers have put a signal peptide on the C- or N-terminus which allowed antibody to penetrate through the cells and still be active. I think that we can produce this to other targets. Cockcroft: What are the kinases that phosphorylate the two residues on Bcl-2? Razin: We don’t know. Nor do we know what is induced by this phosphorylation. We do know that when Hsp90b is going out there is some other protein that takes its place.


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Brown: Do other mast cell survival factors, such as IL3, induce Hsp90? Razin: With IL3 it is the same story. When the cells are driven into the cycle there is association between these two proteins. The dogma that Bcl-2 works as a nude molecule by itself is probably wrong. Reference Fumo G, Akin C, Metcalfe DD, Neckers L 2004 17-Allylamino-17-demethoxygeldanamycin (17AAG) is effective in down-regulating mutated constitutively activated KIT protein in human mast cells. Blood 103:1078–1084

General discussion III

Ono: I’d like to open up this general discussion with a question. Silvia Monticelli, I was thinking about what you said about whether there is an order of appearance of the hypersensitive sites during mast cell development. Your work has been done primarily in mouse, so there is always the question of whether you will see the same sites in human. Dean Metcalfe, have you any cells that might be useful to Silvia? Have you looked at your cell line in terms of its expression of interleukin (IL)13 and IL4, to see whether you can manipulate it pharmacologically so you can influence the induction of such genes? This might be a useful cell reagent for such studies. Metcalfe: We do have a cell line that anyone can request; I do not know about its expression of IL13 and IL4 (Kirshenbaum et al 2003). The cell line is termed LAD2. These cells were cultured from a patient with an aggressive form of mastocytosis. They are stem cell factor (SCF) dependent, and express the high-affinity IgE receptor. In many characteristics they resemble a more mature human mast cell. They grow more slowly and become more granulated than human mast cell line-1 (HMC1) cells. They are not perfect, but are available. The more difficult question relates to examining CD34-derived human mast cells cultured in SCF. This culture system is very complicated. You can’t go in at two weeks and say that all the cells are mast cell precursors because the culture at this point contains diverse cell lineages both dividing and undergoing apoptosis. We have a recent paper illustrating the complexity of these interactions (Kulka & Metcalfe 2005). Brown: Along these lines, a lot of information about T cells and B cells was derived from the study of tumour cell lines that were blocked at certain stages of differentiation. There are a lot of mast cell lines that probably haven’t been characterized because we don’t know about steps in mast cell differentiation. It is something to be thinking about. Rao: We began working with established T cell clones to begin with, and we finally came to the conclusion that the information gained from them wasn’t commensurate with the effort it took to maintain them. Primary cells behave in a reproducible fashion, especially the ones from inbred strains. To address the other point about human and mice, the conservation of hypersensitive sites in the mice suggests that if we did the same assay in the human we would find what we expected. Rivera: It is intriguing that the DNase hypersensitivity sites may be the same between strains of mice, where we know that the genes expressed in these strains 198

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are quite different in many cases. In the Balb/c background we see very high levels of IL2 compared with C57/BL6. This would suggest that what is occurring is regulation at a post-transcriptional level, or that there is some other mechanism going on. Rao: I think DNase I hypersensitive sites appear during cell lineage specification, whereas actual levels of gene transcription by differentiated Th1 and Th2 cells are specified by acute or lineage-specific transcription factors whose expression levels and regulation are subject to control by background-encoded genes. One thing that points to this is that we have made knockouts of the individual hypersensitive sites which either diminish or derepress gene transcription. If we examine the knockout cells, hypersensitivity patterns across the rest of the locus are unchanged. Apparently a cooperative mechanism establishes the open chromatin structure and then acute transcription is modified by signalling mechanisms that act on transcription factors. There are two stages that need to be distinguished. Ono: When I mentioned the human/mouse issue, the data are beautiful. It wasn’t to say that I have any reason to doubt that this is translatable. But especially in the mast cell field it hasn’t always been straightforward to translate cytokine expression in mouse mast cells to human mast cells. There have sometimes been some disagreements. For that reason it would be useful to look at mast cell lines. I have done these DNase hypersensitive site analyses myself, and I realize how many cells are needed. I’m trying to find a way to get enough cells. How many cells would be needed? Monticelli: For our work I like to have 50 million. The minimum needed would be 2 million. References Kirshenbaum AS, Akin C, Wu Y et al 2003 Characterization of novel stem cell factor responsive human mast cell lines LAD1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FceRI or FcgRI. Leukemia Res 27:677–682 Kulka M, Metcalfe DD 2005 High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression. Blood 105:592–599

Mast cells in autoantibody responses and arthritis P. A. Nigrovic*† and D. M. Lee*1 *Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, and †Division of Immunology, Children’s Hospital of Boston, Boston MA, USA

Abstract. A pathogenic role for autoantibodies, immune complexes and mast cells has long been hypothesized in rheumatoid arthritis (RA). Recent studies demonstrating novel RAassociated autoantibodies and the efficacy of B cell-directed therapy have led to a renewed interest in the role of humoral immunity in RA. Mouse models of arthritis have provided further support for a direct pathogenic role of autoantibodies in the development of synovial inflammation. Interestingly, in antibody-mediated K/BxN serum transfer arthritis, mast cells have now been identified as a critical cellular mediator of autoantibody-driven joint inflammation. Here, we focus on the role of autoantibodies and mast cells in murine and human inflammatory arthritis. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 200–214

In addition to their well described role in IgE-driven immune responses and IgEdriven disease states, an expanding literature now documents mast cell participation in diverse immune responses including innate immune functions, induction of adaptive immunity and IgG- and immune complex-dependent disease states. Mast cells express receptors for specific pathogen-associated molecular patterns (PAMPs) such as Toll-like receptors ( TLRs) 1, 2, 4 and 6 and the Gram-negative fibrial protein receptor CD48 (McCurdy et al 2003, Applequist et al 2002, Malaviya et al 1999), thereby enabling potent activation to innate immune stimuli. The importance of mast cell participation in innate immune responses has been underscored by experiments demonstrating that mice lacking mast cells exhibit profound susceptibility to death in certain models of acute bacterial infection (Malaviya et al 1996a). In addition to elaborating chemoattractants that recruit leukocytes to sites of infection, mast cells can participate in induction of adaptive immunity by recruiting lymphocytes to sites of inflammation (Goodarzi et al 2003, Tager et al 2003), by 1

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stimulating dendritic cell and Langerhans cell migration to draining lymph nodes (Bryce et al 2004, Jawdat et al 2004) and by direct antigen presentation (Malaviya et al 1996b). A critical role for mast cells has also been demonstrated in IgG- and immunecomplex driven pathological responses. Mast cells express Fcg R2b and Fcg R3a, low affinity IgG receptors involved principally in the response to immune complexes and other co-localized IgG molecules. Under certain conditions, mast cells may also express the high-affinity IgG receptor Fcg R1 (Okayama et al 2000). In addition, mast cells express receptors for the complement activation fragments C3a and C5a (Nilsson et al 1996). These receptors provide multiple mechanisms by which mast cells can participate in humoral defense and also enable a role for mast cells in antibody- and complement-induced pathology. For example, in the reverse passive Arthus reaction wherein peritonitis is induced by intraperitoneal injection of antibody and systemic (intravenous) injection of antigen, peritoneal mast cells release a burst of pre-formed tumour necrosis factor ( TNF ) and recruit neutrophils (Zhang et al 1992). Similarly, a role for mast cells has been demonstrated in the cutaneous reverse passive Arthus reaction wherein antibody is administered subcutaneously (Zhang et al 1991). Optimal mast cell participation in this cutaneous reaction requires a functional complement system, suggesting that complement fixation by immune complexes provides an important secondary signal to mast cells, in particular via the anaphylatoxin C5a (Ramos et al 1994). A related phenomenon is observed in a model of bullous pemphigoid: subcutaneous administration of an antibody against the hemidesmosomal antigen BP180 induces inflammatory attack, resulting in lysis of the dermal–epidermal junction. In this model of blistering skin disease, absence of mast cells or complement results in dramatic attenuation of inflammation (Chen et al 2001, Liu et al 1995). In addition, synovial mast cell degranulation—a hallmark of cellular activation—has been documented in association with arthritis in several animal models (Gryfe et al 1971, van den Broek et al 1988, Lee et al 2002, CorrCrain 2002). A critical functional role for mast cells in arthritis pathogenesis has recently been firmly established using the autoantibodydependent K/BxN model (Lee et al 2002).

Mast cells in experimental arthritis Autoantibody-dependent arthritis: the K/BxN model The development of the K/BxN mouse model of inflammatory arthritis by Benoist and Mathis (Kouskoff et al 1996) has provided a powerful new approach for investigation of cellular and molecular mechanisms involved in inflammatory synovitis. K/BxN T cell receptor transgenic mice spontaneously develop progressive inflammatory arthritis that displays features of rheumatoid arthritis ( RA) (Kouskoff et al

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1996). These features include synovial hyperplasia, inflammatory cell infiltrates in synovial tissue and synovial fluid, pannus formation and joint destruction (cartilage and bone erosions). Initial studies of the K/BxN transgenic mice revealed that the development of this autoimmune arthritis was dependent on both T and B lymphocytes and MHC class II. However, further analysis revealed that the arthritic phenotype could be conferred on recipient mice, including lymphocyte-deficient recipient mice, by transfer of acellular serum (Korganow et al 1999). The arthritogenic activity of K/BxN serum resides in the IgG fraction and results uniquely from high-titre autoantibodies directed against the autoantigen glucose-6phosphate isomerase (GPI) (Matsumoto et al 1999). Thus, the autoimmune inflammatory arthritis in K/BxN mice results from both lymphocyte-dependent systemic generation of an autoantibody and subsequent lymphocyte-independent synovial effector mechanisms. Immunoglobulin effector functions: complement, IgG FcR, neutrophil and mast cell-dependent arthritis The ability to induce arthritis in recipient mice via passive transfer of pathogenic autoantibodies contained in K/BxN serum (Korganow et al 1999) enables genetic dissection of molecular pathways involved in inflammatory arthritis via analyses in ‘knockout’ mice. Genetic analyses focused on autoantibody-dependent immunopathogenic mechanisms requisite for K/BxN autoantibody-induced synovitis revealed a dependence on both complement and IgG Fc receptor components ( Ji et al 2002). Interestingly, activation of the complement pathway does not require components of the classical pathway (C1 and C4 are dispensable); rather, activation proceeds via the alternative pathway of complement activation (factor B is required). Regarding complement effector function, formation of the MAC complex (utilizing C6–C9) is not required for arthritogenic activity. Instead, induction of arthritis is dependent on interaction of complement split-product C5a (an anaphylatoxin) with its cognate cellular receptor (C5a receptor, CD88) ( Ji et al 2002). Similar analyses revealed a requirement for IgG Fc receptor (FcR): mice lacking the common g chain of the Fc receptor (shared by Fcg RI, Fcg RIII, FceR and others) are resistant to induction of arthritis ( Ji et al 2002). Further analysis demonstrated that the requirement for IgG FcR is dependent on expression of Fcg RIII, while the high-affinity Fcg RI is dispensable ( Ji et al 2002). Since the low affinity but high avidity Fcg RIII is activated optimally by clustered IgG Fc regions in immune complexes which also efficiently activate complement to generate the anaphylatoxin C5a, GPI-anti-GPI immune complex formation appears to be a likely pathogenic event in K/BxN arthritis induction. Immune complexes are further implicated in arthritis pathogenesis by the demonstration of their presence in arthritic synovial fluid and joint surface (Matsumoto et al 2002) and by the


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observation that multiple anti-GPI antibodies with non-overlapping epitope specificities—as would be required to form an antigen-antibody lattice—are required to initiate arthritis (Maccioni et al 2002). Of note, a dependence on complement and stimulation of the Fc receptor has also been demonstrated in the collagen-induced model of inflammatory arthritis (CIA) suggesting that these mechanisms are not unique to the K/BxN model (Diaz de Stahl et al 2002, Watson et al 1987, Wang et al 2000). Thus, the K/BxN model of inflammatory arthritis has demonstrated mechanisms by which systemic autoimmunity driven by lymphocytes and autoantibodies translates into synovial inflammation via IgG, FcR, complement activation and engagement of CD88 (C5aR). An interesting corollary to the genetic requirement for two cellular receptors (C5aR and Fcg RIII) is the implied presence of a responding cellular population within the synovium. Because they are present in abundance in the inflamed synovial tissue and demonstrate evidence of activation (degranulating phenotype) in multiple arthritis models, and because they may possess appropriate activating receptors (Fcg RIII and C5aR) critical for induction of arthritis, synovial mast cells are well positioned to initiate the tissue response to autoantibody-driven K/BxN arthritis. Indeed, analysis of synovial mast cell degranulation demonstrates rapid (within 2 hours) tissue-specific activation of synovial mast cells after administration of arthritogenic K/BxN serum (Lee et al 2002). Further evidence supporting the hypothesis that mast cells participate in K/BxN arthritis pathogenesis derives from mast cell deficient strains of mice: two strains of mice deficient in mast cells (Sl/Sl d and W/W v ) are highly resistant to arthritis (Lee et al 2002). Furthermore, reconstitution of mast cell deficient W/W v mice with normal mast cells restored the wildtype arthritogenic response to administration of K/BxN serum. Taken together, these findings demonstrate a critical role for mast cells in induction of synovial inflammation in the K/BxN model. Although the aforementioned analyses in W/W v mice demonstrate a critical role for mast cells in K/BxN serum transfer arthritis induction, the mechanisms by which mast cells participate in synovial inflammation remain largely undefined. More specifically, mechanisms of synovial mast cell activation are unknown; IgG Fcg RIII and CD88 are prominent candidates. Furthermore, the specific effector functions elaborated by mast cells in arthritis initiation are unknown. Once activated, mast cells in the synovium could initiate inflammation via a number of mechanisms. Inflammatory cytokines, eicosanoids and neutral proteases are produced in great abundance by activated mast cells and have potent pro-inflammatory effects via recruitment of circulating leukocytes and activation of resident synoviocytes. Selected candidate pathways are outlined in Fig. 1. Aside from their role in arthritis induction, the participation of mast cells in perpetuation of chronic arthritis is also unknown. In some murine models of bacterial and antibody-induced disease, the physiological role of mast cells can largely be replaced by a single administra-

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FIG. 1. Candidate pro-inflammatory functions of mast cells in synovitis. Mast cell effector functions suggest their participation in diverse pathogenic pathways in inflammatory arthritis, including leukocyte recruitment and activation, synovial fibroblast activation, hyperplasia, angiogenesis, and cartilage and bone destruction. Activated mast cells elaborate mediators potently capable of enhancing vasopermeability, inducing endothelial expression of adhesion molecules, recruiting circulating leukocytes, and activating infiltrating leukocytes as well as resident macrophages, thereby contributing to the early phases of inflammatory arthritis. In chronic synovitis, mast cells synthesize mitogens and cytokines that activate synovial fibroblasts, recruit macrophages, and promote the growth of new blood vessels, implicating them in synovial lining hyperplasia and pannus formation. Further, mast cells may participate in joint destruction by the induction of matrix metalloproteinases from fibroblasts, by activation of chondrocytes, and by direct and indirect promotion of osteoclast differentiation and activation. Since activated synovial fibroblasts demonstrate enhanced stem cell factor (SCF) expression, a potentially important positive feedback loop is established wherein SCF promotes mast cell survival and proliferation, leading to the mastocytosis described in inflamed synovium. Note that the in vivo importance of these candidate pathways remains to be established. (Graphic design by Steve Moskowitz.) Reproduced with permission from Nigrovic & Lee (2005).

tion of neutrophils or neutrophil chemoattractants (Zhang et al 1992, Echtenacher et al 1996, Malaviya et al 1996b, Chen et al 2001). This observation suggests that mast cells play no substantial ongoing role in these pathologic states. However, in K/BxN mice—which exhibit a progressive erosive arthritis in the setting of persistently high levels of autoantibodies in the serum—ongoing mast cell degranulation is readily evident (Lee et al 2002). This finding suggests that synovial mast cells


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may undergo repetitive cycles of activation in the chronic arthritic state and thus participate in ongoing disease much more substantially than has been observed in models of peritonitis and skin disease. A role for the synovial mast cells beyond the initiation of synovitis in K/BxN arthritis awaits experimental definition. Mast cells in human inflammatory arthritis Histological evidence documenting prominent mast cell infiltrates in the rheumatoid synovium has long implicated this lineage in the pathologic processes functional in inflammatory arthritis. Mast cells are present in limited numbers in normal human synovium. In the context of RA and other inflammatory joint diseases mast cell hyperplasia is frequently noted, with mast cell population expansion to 5% or more of all synovial cells. The number of accumulated mast cells differs substantially from patient to patient, in general varying directly with the intensity of joint inflammation (Crisp et al 1984, Godfrey et al 1984, Gruber et al 1986, Malone et al 1987, Kiener et al 1998, Echtenacher et al 1996, Gotis-Graham et al 1998, Bromley et al 1984, Bromley & Woolley 1984, Tetlow & Woolley 1995). Mast cells are present throughout the inflamed synovial sublining, with occasional microanatomic clustering near sites of cartilage and bone erosion (Bromley & Woolley 1984, Bromley et al 1984). A relative mastocytosis may also be observed in other arthritides, including juvenile rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis and some cases of osteoarthritis (OA) (Godfrey et al 1984). Consistent with their possible role in arthritogenesis, mast cell mediators are present in the synovial fluid of inflamed human joints. These mediators include histamine and tryptase, both considered to be specific for mast cells (Partsch et al 1982, Frewin et al 1986, Malone et al 1986, Buckley et al 1997, Lavery & Lisse 1994). Interestingly, mast cells in RA synovium and not at other anatomic sites have been noted to express CD88, the receptor for the anaphylatoxin complement fragment C5a (Kiener et al 1998). Activation of the complement system is evident in synovial tissues, where complement cleavage products are present at high concentration (Rodman et al 1967, Kinsella et al 1969), as well as in synovial fluid where depressed levels of complement and elevated levels of complement cleavage products have been demonstrated (Ruddy et al 1969, Pekin & Zvaifler 1964). Several studies (Jones et al 1982, Winchester et al 1969, Broder et al 1972) also demonstrate IgG- and complement fragment-containing immune complexes in synovial fluid. Expression of CD88 thus provides a potential mechanism for human synovial mast cells to respond to the activated complement fragments present in synovial fluid and tissues in patients with RA. Although evidence of mast cell activation in human RA joint tissues is readily evident, the mechanisms by which these cells participate in synovial inflammation

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remains a matter of conjecture. Given the expanded numbers of mast cells within the joint and their enormous capacity for production of cytokines and chemokines, it would be surprising if they were of no consequence to the chronic inflammatory response. The broad range of mast cell effector functions includes elaboration of mediators with bioactivity directed at both marrow-derived leukocytes as well as mesenchymal tissue elements (Fig. 1). Both the bone marrow derived and mesenchymally derived lineages display prominent responses in RA. Mast cells are potent sources of chemokines including interleukin (IL)8, MCP-1, MIP-1a and RANTES, which potentially contribute to the extensive leukocyte recruitment evident in RA synovial tissues (Metcalfe et al 1997). Mast cells may also contribute to the activation of these leukocyte populations via production of interferon g and IL6. Since infiltrating leukocytes, and particularly synovial macrophages, are major sources of the pro-inflammatory cytokines TNF and IL1 within the joint, mast cell effects on the size and activation state of the synovial macrophage population may functionally modulate the course of inflammatory arthritis. Mesenchymally derived synovial fibroblasts, which increase greatly in numbers and assume a histological appearance suggestive of increased synthetic activity, are also prominently involved in joint inflammation. Mast cells elaborate an array of mediators with potent effects on synovial fibroblasts including nerve growth factor, basic fibroblast growth factor (bFGF ), platelet-derived growth factor (PDGF ), vascular endothelial growth factor (VEGF ), and transforming growth factor ( TGF )b (LiBaek 2002). Furthermore, mast cell tryptase promotes chemotaxis and collagen synthesis in fibroblasts, while histamine stimulates fibroblast proliferation (Abe et al 2000, Gruber et al 1997, Jordana et al 1988). Interestingly, the communication between mast cells and synovial fibroblasts is bidirectional. Mast cells require stimulation by stem cell factor (SCF ) for in situ differentiation as well as activation (Galli et al 1993). Fibroblasts in inflamed RA synovial tissues express higher levels of SCF, and upregulation of SCF expression has been noted in synovial specimens exposed to TNF (Huttunen et al 2002, Kiener et al 2000, Ceponis et al 1998). Fibroblasts may also promote survival of mast cells via SCF-independent pathways yet to be fully defined (Sellge et al 2004). Conclusions Data from the K/BxN mouse model show that mast cells play a critical role in the pathogenesis of inflammatory arthritis, in particular in arthritis induced by autoantibody-containing immune complexes. Though a similar mechanism remains unproven for human joint inflammation, markers of mast cell activation are observed in joint fluid from patients with chronic arthritis and mast cell numbers are often greatly expanded within the inflamed synovium. While much remains to be learned about the role of the mast cell in arthritis, such a role now appears highly


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likely, offering a potential new target for therapeutic agents in the treatment of RA and other inflammatory diseases of the joints. Acknowledgements Supported by the Physician Scientist Development Award of the Arthritis Foundation and American College of Rheumatology Research and Education Foundation (PAN) and R01-AI059746, K08-AR02214, the Cogan Family Foundation and the Arthritis Investigator Award of the Arthritis Foundation and the American College of Rheumatology Research and Education Foundation.

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DISCUSSION Williams: What happens to neutrophil numbers in the synovium in the mast celldeficient mice? Are the effector cells for the disease really the neutrophils? Do the mast cells produce the signals that bring in more neutrophils, or is it more complex? Lee: It is possible that the role of mast cells is mainly to recruit neutrophils. Unfortunately, one of the limitations of our approach is that it only affords focus on mast cells and responses are binary. Either we get inflammation, in which case there are a lot of neutrophils, or we don’t get much inflammation or neutrophil recruitment. Williams: Do you think neutrophils are important effector cells that produce the clinical symptoms in your model? Are mast cells purely signalling to get neutrophils in by producing neutrophil chemoattractants? Lee: I thought this way for a while, but the situation is more complicated. There is a recent paper by Paul Allen’s group ( Wipke et al 2004) looking at an earlier event (vascular leak—our readout is arthritis at day 14). If one looks within 1 hour of serum administration there is a hyperacute vascular leak in these mice. I would have suspected that this is a result of mast cells releasing an acute inflammatory mediator, but Professor Allen’s work showed a codominant role for neutrophils as well as mast cells even at this early stage. Thus, numerous lineages are participating at different points in this process in a complex interaction. Williams: In other inflammatory models I have looked at in species other than mouse, immune complexes can stimulate the generation of C5a, and C5a can induce neutrophil accumulation directly by acting on C5a receptors on the neutrophil surface. This induces microvascular plasma protein leakage by a mechanism totally dependent on a neutrophil/endothelial cell interaction. Thus, we can bypass the mast cell entirely. Is there something about mice where the C5a receptor on neutrophils is not quite as important and functional as it is in other species? Lee: That’s a great question. Why is the neutrophil which is perfectly competent in many contexts of responding to C5a not just coming in by itself ? It is possible that while the W/W v mouse is capable of recruiting neutrophils to inflammatory sites, the ‘W ’ (c-kit ) mutation may render them less responsive to C5a. I guess I would ask the question back, in that when you did your studies, were mast cells present or absent? Williams: Mast cells were present, but they did not seem to be of central importance in the particular rabbit models and human studies that we investigated. In contrast, in all the mouse models I have seen, the mast cells seem to be pivotal. In rabbits, if we inject C5a or C5a des Arg (which is a poor mast cell activator) into tissues there is neutrophil accumulation and vascular leak, without significant mast cell involvement.


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Lee: My impression was that if you put enough C5a in mice, you have a similar response. Williams: Perhaps ‘enough’ is the critical issue. You may need more C5a in mouse models. You have this amplification provided by mast cells that perhaps is not essential in other similar species. Galli: If mast cells were present in the rabbit, what was the evidence that they weren’t involved? Williams: It was based on mast cell histamine release. Microvascular leak induced by intradermally injected C5a was not suppressed significantly by antihistamines. Furthermore, as I said, C5a des Arg that is a poor mast cell activator was potent at inducing microvascular leakage, and this was totally dependent on neutrophils. This was the major evidence. We looked at other models involving mast cells sensitized with antibodies as a comparison and these were characteristically different. Galli: Sounds like you should definitely stop studying rabbits! Metzger: What are the generic properties of the antigen that initiates this whole process? Lee: That is another compelling question. Why is a glycolytic enzyme an autoantigen for inflammatory arthritis? I have a couple of thoughts. GPI (glucose-6phosphate isomerase) has also been described as displaying cytokine activity. It is present in the circulation at quite high levels in young mice and therefore available to form immune complexes in the vasculature. An intriguing observation in the recent paper by Paul Allen’s group ( Wipke et al 2004) is that when he administers any immune complex or aggregated immunoglobulin he finds that there was a preferential vascular leak in the synovium. Therefore, I am not sure whether the instigating event is a property of the autoantigen or a property of the synovial tissue that is preferentially sensitive to this immune complex and is responding on that basis. Koyasu: You showed that both the C5a receptor and the Fc receptor are required. Do they show a synergistic effect or an independent one? If you transfer the C5a knockout mast cells to an FceRIg knockout mouse, can you induce arthritis? Lee: We haven’t done that, but it is an interesting question. Pecht: If I understand correctly, you always used the whole serum to induce the phenomenon. How much of this effect can you produce by just using the isolated antibodies? Lee: The Mathis and Benoist lab showed that essentially all activity resides in the IgG fraction of serum (Korganow et al 1999, Matsumoto et al 1999). We administer serum as opposed to purified antibody because it’s easier. Rivera: I understand you are now using C57/ BL6 bone marrow-derived mast cells (BMMCs) to reconstitute. One of the issues is the relative ratio of Fcg RIII to Fcg RII on those cells. At least in vitro, they are very difficult to activate with immune complexes. Have you done the synergistic experiment that Shigeo has

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done? This could be done in vitro where you could ask whether C5a is really overcoming that kind of limitation. Lee: Our current ‘holy grail’ is to connect the dots between what we see as an effector function and stimulating specifically by ligation of the Fc receptor and C5a. Others have shown that administration of C5a to macrophages changes the g RII:g RIII ratio. Using the brake/accelerator analogy, perhaps the administration of C5a changes the ratio to more acceleration and less brake. We have tried and have not been able to find this mechanism yet in our in vitro work on BMMCs. Metcalfe: In one of your black box diagrams, just before the black box you had IL1. Is your working hypothesis that IL1 is from mast cells, and if so, what do you know about IL1 synthesis and release from murine mast cells as induced by Fcg RIII or CD88? Lee: Mast cells can make IL1, as assayed by Western blot. Using LPS stimulation as a control in a macrophage control cell line, we are in the same range of IL1 production for BMMCs. One of the problems of ligating Fcg RIII using monoclonal antibodies currently available is that one simultaneously activates both Fcg RIII and inhibitory Fcg RII. We are currently looking at Fcg RII knockout BMMCs and find that ligation of Fcg RIII is quite potent at stimulating IL1b. We are in the middle of trying this on wild-type mast cells. Metcalfe: Have you looked at whether co-ligating through CD88 amplifies the production? Lee: Yes, but we need to repeat the experiment. Galli: You alluded to the issue of the phenotype of the mast cells in the synovium at the time the experiment is performed. This may not be the same as the phenotype of the input population of BMMCs. I agree that there is an interesting parallel between your model involving CD88 and the Fcg RIII and Santa Ono’s model with CCR3 and antibody dependent mast cell activation. It is possible that C5a and IgG1 have synergistic effects on mast cells resident in the joints in vivo but not on BMMCs in vitro. Lee: This is entirely possible. Our hope is that we are going to model this using in vitro BMMC cultures, but it is important to consider BMMCs may not fully model the synovial tissue mast cell. Marshall: Your focus has been on the induction of the arthritis. What about the chronicity of the disease in the response to the drug? Is this consistent? Lee: Since they never develop inflammation, we have not pursued chronicity studies. Marshall: In the situations where you see initial induction, do you see differences in chronicity? Lee: We haven’t taken them out before the full course of antibody transfer. When we can, we have tried administering blocking compounds for arthritis to see if we


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can ameliorate the disease, to see if there is a role in the perpetuation phase. For several of these there is a role. Galli: In your histological studies of joints that are affected or are not affected, have you been able to discern whether there is a substantial expansion of the mast cell population in association with the development of pathology, as compared with baseline levels in normal joints? Lee: The short answer is yes. The problem has been doing true volumetrics in a tissue that has significant anatomic variability. Also the mast cells are not present with a consistent distribution. I can say that if one quantifies total synovial mast cell numbers, there is an increase. One other question we are investigating is the levels of recruitment by looking at progenitor numbers. Galli: Sometimes in these chronic inflammatory models that are associated with tissue remodelling the mast cell population changes in a number of respects, not only in phenotype but also in numbers and anatomical distribution. We have seen in a chronic model of asthma that the mast cell numbers increase two- to threefold in the course of the response. Bradding: Is there much evidence for C5a involvement in human rheumatoid disease? Lee: Yes. If we look at studies from 30 years ago, when aggressive treatments weren’t widespread, we can see high levels of complement and C5a deposition in active synovitis, both in tissue and fluid. Thus, there is evidence for immune complexes and complement activation in inflammatory synovial fluid. Bradding: Have you been able to characterize mast cells in the rheumatoid synovium, in terms of cytokine production and resting number for example? Lee: Others have done this. It is high on the list of things to do for us. If we take human synovium and analyse cytofluorometrically, we find that up to 5% of cells are c-Kit hi e receptor expressing, consistent with a mast cell phenotype. Williams: We did a study 15 years ago measuring C5a levels in synovial effusions from people with rheumatoid arthritis (RA). The levels of C5a were very high. Rivera: Is CD88 expression a phenotype of connective-tissue-type mast cells (CTMCs)? Is expression driven by stem cell factor (SCF)? Lee: There is a paper from Peter Valent’s group (Kiener et al 1998) looking at CD88 expression on synovial mast cell populations. They looked at mast cells from different anatomical sites: osteoarthritic synovium, RA synovium, skin and placenta and found that CD88 was selectively, but not uniquely, expressed on the synovial mast cells from RA. Ono: What happens in juvenile forms of arthritis in the synovium? Lee: I don’t know. Metcalfe: We published a paper on a rat model of arthritis in which we sensitized the synovium with antigen-specific IgE and then administered the antigen system-

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ically (Malone & Metcalfe 1988). In this model we could see swelling of the sensitized joint and extravasation of fluid. If we did it on one occasion there was no residual inflammation. It reminded us of palindromic rheumatism where there aren’t any chronic changes. We were curious about whether if we repeatedly degranulated mast cells we could initiate a chronic process. The facilitation of these processes by mast cells, at least in our way of thinking, seemed to be more by changes in vascular permeability, allowing other cells and molecules to enter. Lee: One of the burning questions regarding the role of mast cells in inflammatory arthritis is do they have an ongoing pathogenic role after disease induction? Mast cells produce high levels of fibroblast mitogens such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). Fibroblast hyperplasia is one of the prominent phenotypes in inflammatory arthritis. Whether or not this is mast cell dependent is unclear. Metcalfe: When we started studying mastocytosis we had an early interest in arthritis. It turns out that the arthritis observed in mastocytosis is usually osteoarthritis and there is no increase in immune arthritis. But when RA did occur in a patient, this arthritis was rapidly destructive. It appeared to us that increasing the effector population of mast cells in the context of autoimmune disease of the joint led to a more aggressive course. Brown: What about multiple sclerosis (MS) in mastocytosis patients? Metcalfe: We have only seen one case of MS in a patient with mastocytosis. This disease appeared no different in its evolution. References Kiener HP, Baghestanian M, Dominkus M et al 1998 Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis. Arthritis Rheum 41:233–245 Korganow AS, Ji H, Mangialaio S et al 1999 From systemic T cell self-reactivity to organ-specific autoimmune disease via immunoglobulins. Immunity 10:451–461 Malone DG, Metcalfe DD 1988 Demonstration and characterization of a transient arthritis in rats following sensitization of synovial mast cells with antigen-specific IgE and parenteral challenge with specific antigen. Arthritis Rheum 31:1063–1067 Matsumoto I, Staub A, Benoist C, Mathis D 1999 Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 286:1732–1735 Wipke BT, Wang Z, Nagengast W, Reichert DE, Allen PM 2004 Staging the initiation of autoantibody-induced arthritis: a critical role for immune complexes. J Immunol 172:7694– 7702

MASTering the immune response: mast cells in autoimmunity Greg D. Gregory, Allison Bickford*, Michaela Robbie-Ryan, Mindy Tanzola and Melissa A. Brown*1 Graduate Program in Immunology and Molecular Pathogenesis, Emory University School of Medicine, Atlanta, GA 30321, and * Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, 320 East Superior Street, Chicago, IL 60611-3010, USA

Abstract. Mast cells are established participants in allergic disease and in protection against extracellular parasites. Recently, it has become apparent that mast cells exert many profound effects on a variety of both innate and adaptive immune responses. Using mast celldeficient WBB6F1/J-kitW/kitWv (W/W v ) mice, we have demonstrated that mast cells are critical for severe disease in a murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). Reconstitution of the mast cell population in the periphery, but not the CNS, restores EAE severity. Mast cells exert their effects at both the inductive and effector phases of disease. EAE is mediated by autoreactive T cells that enter the CNS and initiate inflammatory responses, leading to demyelination within the spinal cord and brain. Although there are no intrinsic defects in W/W v -derived T cells, both CD4+ and CD8+ autoreactive T cell responses are attenuated during early disease in W/W v mice. Thus mast cells are essential for the optimal priming of autoreactive T cells. The entry of encephalitogenic T cells into the CNS is compromised in these mice as well. The effects on early T cell responses are due, in part, to the reduced percentage of activated dendritic cells in the lymph nodes of W/W v mice after disease induction compared with wild-type mice. The finding that mast cells can alter T cell responses in EAE has much broader implications for understanding the impact of these cells on all T cell-mediated responses. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 215–231

Mast cells are aptly called ‘multifunctional effector cells’ (for review see Williams & Galli 2000). Until recently, they were largely ignored outside the realm of allergic research and patient care. The conventional wisdom was that these are ‘end stage’ effector cells in allergic responses. That is, only after the generation of an adaptive immune response that gives rise to allergen-specific T and B cells and the subsequent production of allergen-specific IgE, can mast cells be activated through their 1

This paper was presented at the symposium by Melissa A. Brown to whom correspondence should be addressed. 215

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high affinity Fce receptor (FceRI). However, in recent years an increasing number of Ig-independent agonists have been described including interleukin (IL)3, stem cell factor (SCF), complement components, neuropeptides and microbial products that act through toll-like receptors (TLRs) (Marshall et al 2003, Erdei et al 2004, Hundley et al 2004, Ansel et al 1993). It has also become apparent that, in addition to the well-characterized proteases, vasoactive amines, cysteinyl leukotrienes and prostaglandins released by activated mast cells, mast cells also express a plethora of cytokines and chemokines. The ability of mast cells to be activated prior to Ig production makes it likely that they can act much earlier after immune challenge to influence both adaptive and innate immunity. Our recent work in a murine model of multiple sclerosis (MS) has generated results that support this idea (Secor et al 2000). Here, we review the evidence demonstrating that mast cells are essential for a strong autoreactive T cell response that initiates disease, and that these cells also contribute to the later stages of immune destruction that occur in the CNS. Mast cells, MS and experimental allergic encephalomyelitis MS is a debilitating neurodegenerative disease characterized by local inflammation within the CNS that leads to destruction of the myelin sheath, a structure that protects nerve axons and contributes to nerve conduction (Steinman 1996). Experimental allergic/autoimmune encephalomyelitis (EAE), the rodent model of MS, is similar in many respects to the human disease. However, EAE must be experimentally induced by immunization with myelin-derived peptides (for review see Kuchroo et al 2002). The development of EAE takes place in two temporally distinct stages: the induction and the effector phase (See Fig. 1). In the induction phase, myelin-reactive CD4+ T helper 1 (Th1) cells are activated and undergo expansion in the secondary lymphoid organs. The source of antigen that initiates autoreactive T cell activation in MS is unknown. During the effector phase, self-reactive T cells leave the secondary lymphoid organs and are able to gain access to the CNS through a breach in the relatively impermeable blood brain barrier. Secondary activation of T cells by CNS-derived myelin antigens elicits further T cell proliferation and a cascade of inflammatory responses that involve a variety of other cell types including CD8+ T cells, B cells, macrophages and astrocytes. In 2000, our laboratory reported that mast cells were important determinants of disease severity in EAE. WBB6F1/J-kitW/kitWv mast cell-deficient mice (W/W v ) develop a delayed and milder form of disease than their wild type littermates after immunization with the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)35–55 in complete Freund’s adjuvant (Fig. 2A and Secor et al 2000). These original experiments were based on the hypothesis that local mast cells, normally present in the CNS, were activated during the inflammatory process and contribute to disease. This hypothesis was supported by a variety of data correlating mast cells

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FIG. 1. Experimental allergic encephalomyelitis (EAE), the rodent model of the human demyelinating disease, MS, develops in 2 phases. During the inductive phase, dendritic cells acquire myelin antigen, mature and migrate to peripheral lymph nodes where they activate autoreactive T cells. In the effector phase, antigen specific CD4+ and CD8+ T cells gain entry through the normally impermeable blood–brain barrier (BBB) and initiate a cascade of inflammatory events that cause destruction to the myelin sheath. Potential sites of mast cell influence are shown as well.

FIG. 2. W/W v mice show a delay in disease onset and a reduction in disease severity. (A) Clinical scores were assigned daily to wildtype (n = 16) and W/W v (n = 17) and the mean of each group is reported (P < 0.0001) as determined by the paired t-test. (B) Reconstitution of W/W v mice with bone marrow-derived mast cells (BMMCs) restores EAE disease severity to wildtype levels. Clinical scores were assigned daily to wildtype (n = 10) and W/W v (n = 8) and wildtype +BMMCs (n = 12). Reproduced with permission from Secor et al (2000).

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with disease. For example, many mediators implicated in the myelin damage and axonal transection that leads to loss of nerve conduction are produced by activated mast cells (Steinman 1996, Bjartmar et al 2003, Gordon et al 1990). Among these are nitric oxide, oxygen radicals, tumour necrosis factor ( TNF )a and proteases. Mast cells are a predominant source of vasoactive amines such as histamine, a molecule that can influence blood–brain barrier (BBB) permeability ( Theoharides 1990). Although many of these molecules could be acting systemically (via increased serum levels), evidence also exists that mast cells exert an influence locally within the CNS. Degranulated mast cells are associated with MS plaques, and transcripts of mast cell-specific genes (e.g. tryptase and FceRI) are increased in lesions of patients with both acute and silent disease compared to normal control subjects (Toms et al 1990, Lock et al 2002). However, the results of our subsequent studies revealed an additional role for mast cells in the disease process. Mast cells influence disease at sites outside the CNS The mast cell defect in W/W v mice can be corrected by selectively reconstituting with wild-type bone marrow-derived mast cells (BMMCs) (Galli & Kitamura 1987). If mast cell repletion restores the wild type phenotype in a disease setting or immune response, this provides good evidence of mast cell participation. Virtually pure BMMC precursors are introduced systemically into mice intravenously or locally via injection at specific sites. Mature mast cell populations are restored to many normal sites within 8–10 weeks. In the EAE model, mast cell reconstitution results in a restoration of disease severity to wild-type levels (Fig. 2B and Secor et al 2000). Surprisingly, this occurs despite the fact that the transferred mast cells do not appreciably populate the CNS (Fig. 3), even after disease induction ( Tanzola et al 2003). The lack of mast cells at sites of inflammatory CNS damage, in the face of severe disease, required reassessment of the possible modes of mast cell influence. Mast cells are widely distributed throughout the body at sites that include the skin, connective tissues and mucosa of the respiratory and gastrointestinal tract ( Williams & Galli 2000). Of particular interest was the observation that mast cells are also normally present in the lymph node and spleen ( Tanzola et al 2003). These locations are important sites for the generation of antigen-specific T cells. Thus, we reasoned that perhaps mast cells are acting not only in the CNS during the effector phase of EAE, but also much earlier, in the inductive phase of disease when T cell priming occurs. W/W v derived T cells are not inherently defective In studies of T cell function in W/W v mice, it was important for us first to establish that their T cells have no inherent defects. The mast cell defect in these mice


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FIG. 3. Comparison of mast cell distribution in the CNS and inguinal lymph node in wild-type, BMMC-reconstituted and bone marrow-reconstituted W/W v mice. Shown by toluidine blue staining, mast cells (arrows directed to representative mast cells) are present in wildtype mice in the meninges around the spinal cord (a) and in the inguinal lymph node (d). (b, e) BMMC reconstitution does not restore mast cells to the CNS (spinal cord meninges shown here) or lymph node of W/W v mice. (c, f ) Transfer of whole bone marrow restores mast cells to the W/W v spinal cord meninges and inguinal lymph nodes after 12 weeks. Reproduced with permission from Tanzola et al (2003).

is due to two distinct naturally occurring mutations in the SCF receptor, c-Kit, which result in a 90% reduction of signalling activity (Nocka et al 1990). Mast cells are exquisitely dependent on SCF for normal development and thus W/W v mice virtually lack mast cells. However, deficiencies in c-kit activity conferred by the W/W v mutations have the potential to affect the development of several haematopoietic cell lineages. This is evidenced in ‘vickid’ mice (viable c-Kitdeficient), which have a distinct c-Kit mutation resulting in defective T cell development (Waskow et al 2002). A scenario in which T cells are defective in W/W v mice could partially account for the reduced disease course observed. To test this possibility, equal numbers of naïve T cells from wild-type and W/W v mice were transferred to ab T cell-deficient mice (TCRb -/-). The next day, animals were immunized with MOG35–55 and disease severity was scored over time. Both groups of animals exhibited equivalent disease kinetics and disease severity (M. Robbie-Ryan,

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data not shown). These data confirm that there is no intrinsic defect in W/W v derived T cells and, if primed in a mast cell ‘sufficient’ environment (in this scenario, the TCRb-/- mouse), they are indistinguishable from wild-type cells in their disease-inducing ability. Mast cells are necessary for optimal priming of autoreactive T cells We next assessed the autoreactive T cell responses in wild-type and W/W v mice. T cells were examined directly ex vivo at early time points post disease induction, prior to any clinical signs of disease. In these experiments, the T cells are ‘primed’ in their native wild-type or mast cell-deficient (W/W v ) environment. Lymph nodes and spleens from immunized mice were harvested at days 8, 11, 13 and 20. Indices of T cell activation were assessed by flow cytometric analysis of the cell surface expression of hallmark activation markers, such as CD11a and CD44. Ex vivo expression of interferon (IFN)g, a cytokine produced by activated Th1 cells, was also determined using intracellular cytokine staining. The expression of CD11a and CD44 is significantly reduced in CD4+ T cells from W/W v mice when compared to wild type cells, as is IFNg production (M. Robbie-Ryan, data not shown). Although MS is thought to be dependent on CD4+ T cells, recent studies provide evidence that CD8+ T cells also play a role in the pathology of disease (Steinman 2001). MOG35–55 contains the MHC Class I restricted epitope 37–46 which elicits a strong CD8+ T cell response (Sun et al 2001, FordEvavold 2005). These MOGspecific T cells can mediate disease alone when transferred to naïve hosts (FordEvavold 2005). Surprisingly, CD8+ T cell responses are affected more profoundly by mast cell deficiencies than T helper responses. The relative expression of activation markers and IFNg is lower in CD8+ T cells isolated from W/W v mice than those from wild type animals (M. Robbie-Ryan and G. Gregory, unpublished data). Such attenuation of CD8+ T cell responses may result from direct requirement of mast cell-derived signals or could reflect the need for a fully functional T helper cell response. Dendritic cell function is compromised in W/W v mice Taken together, these data establish that the initial autoreactive CD4+ and CD8+ T cell response is sub-optimal in W/W v mice and this difference is not due to intrinsic T cell defects. How do mast cells influence very early T cell responses? There are at least two plausible explanations: first, although best known for their expression of Th2 cytokines, mast cells are also important producers of other mediators that influence Th1 differentiation including IL12 and IFNg ( Williams & Galli 2000). Thus, mast cells in the lymph nodes and spleen may act directly on the newly primed T cell to amplify polarized differentiation. Alternatively, the activation of antigen


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presenting cells (APCs) may be dependent on mast cells. Dendritic cells are APCs that are required for the initial activation and proliferation of antigen-specific T cells (Maldonado-Lopez et al 2001, Martin-Fontecha et al 2003). Immature dendritic cells residing in peripheral sites such as the skin are poised to acquire and process antigen followed by migration to the secondary lymphoid organs for presentation to T cells. This maturation and migration is dependent on the activation of dendritic cells by a variety of stimuli such as co-stimulatory molecules, bacterial-derived components and cytokines such as TNFa. It is probably not just a coincidence that mast cells are located in tissues near sites where dendritic cells reside. Mast cells also secrete mediators including TNFa, IL4, GM-CSF and histamine that are known to regulate dendritic cell maturation or migration (Maldonado-Lopez et al 2001). Functionally distinct populations of dendritic cells have been described that can differentially influence the migration and T helper cell fate (Maldonado-Lopez et al 2001). Our preliminary data suggest that while there are no differences in bone marrow derived dendritic cells differentiated in vitro, the numbers of both CD8a+ and CD8a- subsets is diminished in lymph nodes of W/W v mice at day 8 postimmunization with MOG35–55 (Fig. 4 and A. Bickford, unpublished observations). Thus, an attenuated T cell response in W/W v mice may result from less efficient T cell activation due to fewer APCs available and less co-stimulatory signals delivered. A combination of both scenarios may also be possible. The direct effects of mast cells within the lymph nodes on these processes have not been examined.

FIG. 4. Reduced numbers of CD11c+ cells (enriched for dendritic cells) are present in the lymph nodes of mast cell deficient mice after immune challenge. CD8a expression correlates with increased ability to induce Th1 responses (Maldonado-Lopez et al 2001). Single cell preparations of lymph node cells were harvested from wild-type and W/W v mice on day 8 post-immunization and analysed for the expression of CD11c+ and CD8a.

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A role for mast cells in the CNS? A key checkpoint in the progression of MS is the entry of autoreactive cells into the CNS where they re-encounter myelin antigens and undergo secondary activation. The BBB, vascular endothelium characterized by tight cell junctions, is relatively impermeable to the passage of large molecules and cells. However, under inflammatory conditions, a breach in the BBB allows the influx of T cells and granulocytes. Histamine and serotonin, both produced by mast cells, can alter BBB permeability (Repka-Ramirez & Baraniuk 2002, Theoharides 1990) and we have postulated that this is one function of mast cells in disease. Chemokines produced by resident CNS mast cells may also contribute to the directed migration of inflammatory autoreactive T cells to this site. As a first step in evaluating this possibility, we determined the presence of T cells that are in the CNS at day 21 post-immunization. Wild-type and W/W v mice were perfused and flow cytometric analyses were performed to identify activated T cells in the brain and spinal cord. As shown in Fig. 5, the numbers and activation state, as measured by expression of CD25, of CD4+ and CD8+ T cells present in the spinal cord and brain were reduced in immunized W/W v mice. The reduction is most striking in the CD8+ T cell population. This deficit in CD4+ CNS T cells can be overcome by reconstitution with wildtype cells, whereas CD8+ T cell recruitment is not (G. Gregory, unpublished observation). It is possible that mast cell mediators, acting within the CNS, are critical for attracting CD8+ T cells. Leukotriene B4 (LTB4) was recently shown to recruit CD8+ T cells to sites of inflammation and mast cells are a primary source of LTB4 in vivo (Ott et al 2003, Tager et al 2003, Goodarzi et al 2003). The possibility that LTB4-producing mast cells contribute to CNS trafficking is thought provoking. However, difficulties involved in specifically reconstituting the mast cell CNS compartment must be overcome in order to test

FIG. 5. The brain and spinal cord of W/W v-derived mice have fewer activated T cells, as measured by expression of CD25, on day 21 post-immunization. Flow cytometric analysis of CD25 expression on CD4+ and CD8+ T cells isolated from wild type and W/W v mice.


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this hypothesis. While, it is assumed that the differences in cell numbers reflect efficiency of cell entry into the CNS, we cannot rule out the possibility that there is differential expansion of cells within the CNS. Of interest, LTB4 is found at elevated levels in the CSF of MS patients and treatment of mice with an LTB4 inhibitor (CP-105,696) significantly delayed disease onset and reduced disease severity (Neu et al 2002, Gladue et al 1996). How are mast cells activated during disease? Our work has established that mast cells impact the disease course of EAE through effects on T cells and dendritic cells. Yet how mast cells are activated in vivo and which mast cell mediators are relevant in the disease process is still largely unknown. The ability to reconstitute W/W v mice with in vitro-derived mast cells was exploited in studies to determine whether signalling occurs through the Fc receptors on mast cells (Robbie-Ryan et al 2003). W/W v mice were reconstituted with mast cells derived from mice with targeted deletions within individual genes encoding activating and inhibitory Fc receptors. W/W v mice reconstituted with Fcg RI/III-/mast cells (the activating receptor) have less severe disease than those reconstituted with wildtype mast cells, whilst reconstitution with Fcg RIIB-/- mast cells (the inhibitory receptor) results in increased disease severity. These data indicate that myelin-specific antibodies, present in both murine and human disease (Cross et al 2001), modulate mast cell activation through specific Fc receptors. Because mast cells also contribute to events occurring during disease induction, prior to antibody production, we speculate that other signals activate mast cells early in disease. The use of genetically deficient mast cells in reconstitution experiments is a powerful strategy to identify other signalling molecules as well as mediators that regulate disease severity and progression. Summary The finding that mast cells can alter T cell responses in EAE has much broader implications. These findings clearly establish mast cells as important contributors to an optimal T cell response. They act at the earliest stages of antigen encounter, but also influence the effector phase of disease. An additional complexity in the analysis of mast cell influence on disease that must be considered is the observation that there are profound strain specific differences in mast cell mediator production (G. Gregory, unpublished observations, and Bebo et al 1996). Such distinctions could alter the consequences of mast cell influence on adaptive responses and innate responses depending on the genetic background of the individual. Despite such complexity, it is clear that mast cells are no longer solely of interest to allergists. A complete understanding of these potent,

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yet still under appreciated cells will most certainly lead to important advances in the therapy of many diseases. References Ansel J, Brown JR, Payan DG, Brown MA 1993 Substance P selectively activates TNF-alpha gene expression in murine mast cells. J Immunol 150:4478– 4485 Bebo BF Jr, Lee CH, Orr EL, Linthicum DS 1996 Mast cell-derived histamine and tumor necrosis factor: Differences between SJL/J and BALB/c inbred strains of mice. Immunol Cell Biol 74:225–230 Bjartmar C, Wujek JR, Trapp BD 2003 Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease. J Neurol Sci 206:165–171 Cross AH, Trotter JL, Lyons J 2001 B cells and antibodies in CNS demyelinating disease. J Neuroimmunol 112:1–14 Erdei A, Andrasfalvy M, Peterfy H, Toth G, Pecht I 2004 Regulation of mast cell activation by complement-derived peptides. Immunol Lett 92:39– 42 Ford ML, Evavold BD 2005 Specificity, magnitude, and kinetics of MOG-specific CD8+ T cell responses during experimental autoimmune encephalomyelitis. Eur J Immunol 35:76–85 Galli SJ, Kitamura Y 1987 Genetically mast-cell-deficient W/Wv and Sl/Sld mice: their value for the analysis of the roles of mast cells in biologic responses in vivo. Am J Pathology 127:191–198 Gladue RP, Carroll LA, Milici AJ et al 1996 Inhibition of leukotriene B4-receptor interaction suppresses eosinophil infiltration and disease pathology in a murine model of experimental allergic encephalomyelitis. J Exp Med 183:1893–1898 Goodarzi K, Goodarzi M, Tager AM, Luster AD, von Andrian UH 2003 Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues. Nat Immunol 4:965–973 Gordon J, Burd P, Galli SJ 1990 Mast cells as a source of multifunctional cytokines. Immunol Today 11:458– 464 Hundley TR, Gilfillan AM, Tkaczyk C et al 2004 Kit and FcepsilonRI mediate unique and convergent signals for release of inflammatory mediators from human mast cells. Blood 104:2410–2417 Kuchroo VK, Anderson AC, Waldner H et al 2002 T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Annu Rev Immunol 20:101–123 Lock C, Hermans G, Pedotti R et al 2002 Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 8:500–508 Maldonado-Lopez R, Maliszewski C, Urbain J, Moser M 2001 Cytokines regulate the capacity of CD8alpha(+) and CD8alpha(-) dendritic cells to prime Th1/Th2 cells in vivo. J Immunol 167:4345– 4350 Marshall JS, McCurdy JD, Olynych T 2003 Toll-like receptor-mediated activation of mast cells: implications for allergic disease? Int Arch Allergy Immunol 132:87–97 MartIn-Fontecha A, Sebastiani S, Hopken UE et al 2003 Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J Exp Med 198:615–621 Neu IS, Metzger G, Zschocke J, Zelezny R, Mayatepek E 2002 Leukotrienes in patients with clinically active multiple sclerosis. Acta Neurol Scand 105:63–66 Nocka K, Tan JC, Chiu E et al 1990 Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W. EMBO J 9: 1805–1813 Ott VL, Cambier JC, Kappler J, Marrack P, Swanson BJ 2003 Mast cell-dependent migration of effector CD8+ T cells through production of leukotriene B4. Nat Immunol 4:974–981


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Repka-Ramirez MS, Baraniuk JN 2002 Histamine in health and disease. Clin Allergy Immunol 17:1–25 Robbie-Ryan M, Tanzola MB, Secor VH et al 2003 Cutting edge: both activating and inhibitory Fc receptors expressed on mast cells regulate experimental allergic encephalomyelitis disease severity. J Immunol 170:1630–1634 Secor VH, Secor WE, Gutekunst CA, Brown MA 2000 Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 191:813–822 Steinman L 1996 Multiple sclerosis: A coordinated immunological attack against myelin in the central nervous system. Cell 85:299–302 Steinman L 2001 Myelin-specific CD8 T cells in the pathogenesis of experimental allergic encephalitis and multiple sclerosis. J Exp Med 194:F27–F30 Sun D, Whitaker JN, Huang Z et al 2001 Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice. J Immunol 166:7579–7587 Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD 2003 Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat Immunol 4:982–990 Tanzola MB, Robbie-Ryan M, Gutekunst CA, Brown MA 2003 Mast cells exert effects outside the central nervous system to influence experimental allergic encephalomyelitis disease course. J Immunol 171:4385– 4391 Theoharides TC 1990 Mast cells: the immune gate to the brain. Life Sci 46:607–617 Toms R, Weiner HL, Johnson D 1990 Identification of IgE-positive cells and mast cells in frozen sections of multiple sclerosis brains. J Neuroimmunol 30:169–177 Waskow C, Paul S, Haller C, Gassman M, Rodewald H 2002 Viable c-Kit(W/W) mutants reveal pivotal role for c-kit in the maintenance of lymphopoiesis. Immunity 17:277–288 Williams CM, Galli SJ 2000 The diverse potential effector and immunoregulatory roles of mast cells in allergic disease. J Allergy Clin Immunol 105:847–859

DISCUSSION Koyasu: You pointed out many possible interactions between mast cells, dendritic cells and T cells. You used the tumour necrosis factor-a (TNFa) knockout mast cells for the reconstitution experiments. What happens with dendritic cell migration? Brown: We haven’t looked at that. Koyasu: Mast cells are class II-positive and can also present antigens to T cells. Have you used class II knockout mast cells in your reconstitution experiments? Brown: We haven’t done that. We have not been able to demonstrate class II on mast cells in vivo. Has anyone? There are reports in vitro. Koyasu: In inflamed lymph node you may be able to induce class II. Brown: We are able to look in spleen, and get enough cells to analyse markers on mast cells by flow cytometry. We have not seen class II, but we have not looked in lymph node because we haven’t been able to get the numbers needed for reliable results. Galli: Other groups, including my own, have had difficulty finding substantial levels of class II expression on mast cells in vivo. Stevens: It appears that some mast cell populations are relatively good antigenpresenting cells whereas others are not. The human leukaemia mast cell line HMC1 expresses MHC class II and can present a Staphylococcal antigen efficiently to a

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CD4+ T cell hybridoma (Dimitriadou et al 1998, Poncet et al 1999). Cord bloodderived human mast cells also express class II, and exposure to IL4, GM-CSF and IFNg results in increased expression of class II (Love et al 1996, Grabbe et al 1997). Rat peritoneal mast cells constitutively express MHC class II and also are able to present antigen (Fox et al 1994). As occurs in human mast cells, the levels of class II also increase when rat peritoneal mast cells are exposed ex vivo to interferon (IFN)g for 48 hours (Warbrick et al 1995). With regard to the mouse, Frandji et al (1995) reported that IL4/GM-CSF/IFNg -developed mouse bone marrow-derived mast cells (mBMMCs) express class II and can present antigens. However, these investigators also reported that IL3-developed mBMMCs cannot present antigen effectively because IL3 down-regulates Ia expression. Based on the accumulated data, the mast cell’s tissue microenvironment appears to be the primary factor that determines whether or not this immune cell can present antigen effectively. A contributing factor appears to be the state of activation of the cell because it has been reported that class II molecules tend to accumulate in the granule and only reach the plasma membrane in abundance when the cell is cytokine- or FceRI-activated (Vincent-Schneider et al 2001, Tkaczyk et al 1999). Galli: John Schrader showed long ago that mouse mast cell populations generated in vitro expressed class II after stimulation with g interferon (Koch et al 1984). Stevens: In the recovery phase of a helminth infection, most unwanted jejunal mast cells and eosinophils translocate to the draining lymph nodes (Friend et al 2000). Unlike senescent eosinophils that preferentially are retained at this site, most of the senescent mast cells slowly make their way into the blood. Eventually, they are found in the spleen which is the primary graveyard for hematopoietic cells. Dr McNeil’s group also found that cutaneous mast cells translocate to the regional lymph nodes and then the spleen in a dinitrofluorobenzene-induced mouse model of contact hypersensitivity (Wang et al 1998). Confirmatory follow-up studies were carried out in the Wang et al (1998) study using mice that received labelled mBMMCs. The in vitro-differentiated cells were injected in the mouse’s footpads, the footpads were treated with dinitrofluorobenzene, and then the movement of the cells to the lymph node and spleen was measured. Brown: How did he assay that? Stevens: The mBMMCs used in the Wang et al study were tagged with a fluorescent marker. In our study, we confirmed our conclusion that jejunal mast cells can translocate to the spleen by monitoring the movement of v-abl expressing V3 mast cells from the peritoneal cavity to the spleen using an anti-v-abl antibody. Brown: We have tried to look for class II. We have seen other activation markers such as CD44 on mast cells. Koyasu: When the tissue mast cells move to the draining lymph node, what chemokine is used? Usually, CCL7 is a critical chemokine receptor for dendritic cells and T cells.


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Brown: I don’t know. I think MIP1a is for T cells. Galli: You indicated that the deficit in trafficking of T cells into the brain and spinal cord observed in W/W v mice was repaired when you did mast cell engraftment from the congeneic normal mice. Were the phenotypic differences in T cells, and trafficking to lymph nodes, that you observed during the course of disease in W/W v versus +/+ mice also repaired in the mast cell engrafted W/W v mice? Brown: We have not been able to do those experiments successfully. Since we moved the batch of MOG we have is killing our animals within two days. Galli: I know another investigator who has suddenly identified difficulties with preparations of MOG. Perhaps you are getting it from the same source. Was it that you were unable to test the mast cell reconstituted mice? You have identified a difference in T cell phenotype and trafficking to lymph nodes that seems to be Kit dependent. The next step is to assess whether it is mast cell dependent. Brown: We had two experiments where we looked at trafficking at day 21. The experiments where we tried to look earlier were initiated with the bad batch of MOG. Stevens: As previously mentioned, most unwanted jejunal mast cells and eosinophils in the recovery phase of a helminth infection translocate to the draining lymph node (Friend et al 2000). While most eosinophils remain at that site, the senescent mast cells do not. Thus, have you carried out a kinetic experiment to evaluate when mast cells enter the lymph node and how long they stay at that site? Brown: The only experiments we have done looking at mast cell movement into the lymph nodes are in the context of our reconstitution experiments where we transferred GFP-expressing mast cells intravenously. We then looked at almost weekly intervals up to week 28 for mast cells. We never saw them in the lymph node, but we saw them in the spleen. The only time we could demonstrate mast cells in the lymph node was after immunization at about day 24. The animals were sick by then. We didn’t see them earlier. Stevens: Most mBMMCs will quickly move to the spleen when they are injected in a mouse’s tail vein. If I understand your presentation, you are finding in your model that many of these cells eventually appear in the lymph node. Brown: Not mysteriously. I assume that the process of immunization and the inflammatory signals that are generated during the immunization are directing movement of these cells to and from the secondary lymphoid organs. Stevens: Are the mast cells exiting the spleen in your model? Brown: There are data consistent with that idea because the numbers in the spleen drop quite dramatically at the same time we see increases in the lymph nodes. Mast cells are normal residents of lymph nodes, and in wildtype animals we see them easily. They might already be there, and probably are already there in the wildtype animal.

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Rivera: In the TNF knockout reconstitution, do you see the same repopulation patterns? Brown: No, we see them in the spleen. We don’t see them in the lymph node with immunization. Lee: Are you injecting BMMCs into mice and looking 21 days after tail vein injection? Or is there 8 week engraftment and then 21 days? Brown: We have 8–10 week engraftment and then look 21 days later. Lee: At 21 days some other progenitor could come up. Marshall: To what extent is the response dependent on the use of Pertussis toxin as an adjuvant? Brown: I don’t think it is dependent on Pertussis toxin because we have this result in two experiments without it. It is dependent on complete Freund’s adjuvant (CFA), and we have shown that mycobacterium used in the CFA can directly activate the mast cells as assayed by cytokine production. In our model this is an important component. Metcalfe: One of the areas we don’t know much about in mast cell biology concerns the function of mast cells in the brain. You tried to reconstitute mast cells in the brain. Can you elaborate on this? I think of mast cells in the brain as located around vessels. When you look to see reconstitution in the normal mouse, where do you see mast cells in the brain? Brown: The mast cells are in the parenchyma of the brain, but they are also concentrated perivascularly. They lie along the spinal cord and are quite easy to find. When we reconstituted we didn’t see them. Metcalfe: What is the density of the mast cells in the brain? Brown: I couldn’t answer this. Galli: Dr Kitamura has done a mapping of mast cells in the mouse brain. Kitamura: There are a very small number of mast cells in the brain. In contrast, cultured mast cells localized in the spleen of W/W v mice was much higher than normal mast cell number. There are some data that mast cells in the lymphoid organ are very important in the reconstitution of the cultured mast cells in the W/W v mouse. Your data suggested that cultured mast cells localized in lymphoid organ of W/W v mice played very important roles. Ono: It was striking that you saw a decrease in the number of CD8+ T cells in CNS. In those animals if you look earlier at the node, what do the T cells look like? Do they appear to be apoptosing? Brown: No, they don’t look like they are undergoing apoptosis. My interpretation is that there is just not the normal expansion of CD8+ T cells. Ono: Have you looked at the peptide specificity of the CD8+ T cells that make it to the CNS? Brown: No, but MOG35–55 contains both a CD4+ and CD8+ epitope, so it wasn’t surprising that we got a good CD8 response with that immunization. We have


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looked at the antigen-specific cells that are making IFNg after in vitro restimulation with MOG peptide before we assay for cytokines. That’s all we have done so far. Ono: I wonder whether the impact of the mast cell in the node might be somehow affecting the fidelity of the response of the T cells in the node. Lee: There were a couple of papers showing the role of mast cell LTB4 production in CD8+ T cell trafficking (Ott et al 2003, Tager et al 2003). Brown: That is a possible candidate. Both tryptase and LTB4 are found in quite high levels in the cerebrospinal fluid of MS patients (Rozniecki et al 1995, Neu et al 2002). Marshall: One possibility would be CXCR3 ligands. There is strong evidence for them being important in that process. Mast cells can be a potent source of these chemokines. Galli: When Rosetta Pedotti was working with Larry Steinman and me, she showed that MOG-induced experimental allergic encephalomyelitis (EAE) was markedly diminished in mice which genetically lacked the FcR g chain and therefore could bind neither IgG1 nor IgE via Fcg RIII or FceRI (Lock et al 2002) and in FcgRIII knock out mice (Pedotti et al 2003). This conclusion was based on assessment of clinical score and death rates. It is simpler for me to think of those results as reflecting an effector function of the Fcg RIII receptor, rather than an immunoregulatory function. But perhaps this is a complicated system in which there are some effects of FcRg chain signalling on the evolution of the immune response and some on the effector arm of the response. The latter effects are difficult to study in the mast cell engrafted W/W v mice because the transferred mast cells apparently don’t get into the CNS. Brown: We showed the same thing with the Fcg RIII-deficient mice. Stevens: Drs Mekori & Metcalfe (1999) and others (Castells et al 2001) showed that mast cells and T cells can form physical contacts at least in vitro. When mast cells are activated, transmembrane tryptase (TMT)/tryptase g reaches the outer plasma membrane (Wong et al 2002). We showed in our 2002 study that recombinant TMT can induce cultured Jurkat T cells to increase their expression of hundreds of genes. If mast cells physically contact T cells in your EAE model, it is possible that TMT participates in this disorder. TMT is expressed in mast cells in a strain-dependent manner (Wong et al 2002). For example, BALB/c mBMMCs differ from C57BL/6 mBMMCs in their mTMT expression. Thus, is the pathology different in these mouse strains? Because you don’t see mast cells in the brain in your EAE model, an alternate possibility is that an exocytosed mediator from a mast cell that resides in a different organ can make its way into the brain of the EAEtreated animal. Some exocytosed mast cell mediators can exert their effects at distinct sites. An example is mMCP-7. When systemic anaphylaxis is induced in the V3 mastocytosis mouse, some exocytosed mMCP-7 is able to reach the peripheral blood (Ghildyal et al 1996). No inhibitor has been identified that can inactivate

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mMCP-7, and mMCP-7 circulates in the blood for up to 4 hours as a homotypic tetramer. In humans that have undergone a systemic anaphylaxis reaction, a substantial amount of tryptase b also gets into the circulation. That is why tryptase b radioimmunoassays are used in the clinic to identify patients who have undergone systemic anaphylaxis (Enander et al 1991). Have you ruled out the possibility that a mast cell-derived mediator released in the lymph node or spleen is able to get in the brain? Brown: Our reconstitution experiments would support this idea. We are reconstituting the animals, the cells are not getting to the brain and yet we can restore trafficking to the brain through this reconstitution. This supports the idea that something is working distally. Galli: When Rosetta Pedotti performed a microarray analysis of acute, proteolipid protein (PLP)-induced EAE in mice in a collaboration between Larry Steinman’s group and mine, she identified mMCP-7 as one of the up-regulated genes in both brain and spinal cord (Pedotti et al 2003). References Castells MC, Klickstein LB, Hassani K 2001 gp49B1-alpha(v)beta3 interaction inhibits antigeninduced mast cell activation. Nat Immunol 2:436– 442 Dimitriadou V, Mecheri S, Koutsilieris M, Fraser W, Al-Daccak R, Mourad W 1998 Expression of functional major histocompatibility complex class II molecules on HMC-1 human mast cells. J Leukoc Biol 64:791–799 Enander I, Matsson P, Nystrand J et al 1991 A new radioimmunoassay for human mast cell tryptase using monoclonal antibodies. J Immunol Methods 138:39–46 Fox CC, Jewell SD, Whitacre CC 1994 Rat peritoneal mast cells present antigen to a PPDspecific T cell line. Cell Immunol 158:253–264 Frandji P, Tkaczyk C, Oskeritzian C et al 1995 Presentation of soluble antigens by mast cells: upregulation by interleukin-4 and granulocyte/macrophage colony-stimulating factor and downregulation by interferon-gamma. Cell Immunol 163:37–46 Friend DS, Gurish MF, Austen KF, Hunt J, Stevens RL 2000 Senescent jejunal mast cells and eosinophils in the mouse preferentially translocate to the spleen and draining lymph node, respectively, during the recovery phase of helminth infection. J Immunol 165:344–352 Ghildyal N, Friend DS, Stevens RL et al 1996 Fate of two mast cell tryptases in V3 mastocytosis and normal BALB/c mice undergoing passive systemic anaphylaxis: prolonged retention of exocytosed mMCP-6 in connective tissues, and rapid accumulation of enzymatically active mMCP-7 in the blood. J Exp Med 184:1061–1073 Grabbe J, Karau L, Welker P, Ziegler A, Henz BM 1997 Induction of MHC class II antigen expression on human HMC-1 mast cells. J Dermatol Sci 16:67–73 Koch N, Wong GH, Schrader JW 1984 Ia antigens and associated invariant chain are induced simultaneously in lines of T-dependent mast cells by recombinant interferon-gamma. J Immunol 132:1361–1369 Lock C, Hermans G, Pedotti R et al 2002 Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 8:500–508 Love KS, Lakshmanan RR, Butterfield JH, Fox CC 1996 IFN-gamma-stimulated enhancement of MHC class II antigen expression by the human mast cell line HMC-1. Cell Immunol 170:85–90


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Mekori YA, Metcalfe DD 1999 Mast cell–T cell interactions. J Allergy Clin Immunol 104:517–523 Neu IS, Metzger G, Zschocke J, Zelezny R, Mayatepek E 2002 Leukotrienes in patients with clinically active multiple sclerosis. Acta Neurol Scand 105:63–66 Ott VL, Cambier JC, Kappler J, Marrack P, Swanson BJ et al 2003 Mast cell-dependent migration of effector CD8+ T cells through production of leukotriene B4. Nat Immunol 4:974–981 Pedotti R, DeVoss JJ, Youssef S et al 2003 Multiple elements of the allergic arm of the immune response modulate autoimmune demyelination. Proc Natl Acad Sci USA 100:1867–1872 Poncet P, Arock M, David B 1999 MHC class II-dependent activation of CD4+ T cell hybridomas by human mast cells through superantigen presentation. J Leukoc Biol 66:105–112 Rozniecki JJ, Hauser SL, Strein M, Lincoln R, Theoharides TC 1995 Elevated mast cell tryptase in cerebrospinal fluid of multiple sclerosis patients. Ann Neurol 37:63–66 Tager AM, Bromley SK, Medoff BD et al 2003 Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat Immunol 4:982–990 Tkaczyk C, Villa I, Peronet R, David B, Mecheri S 1999 FceRI-mediated antigen endocytosis turns interferon-g-treated mouse mast cells from inefficient into potent antigen-presenting cells. Immunology 97:333–340 Vincent-Schneider H, Thery C, Mazzeo D, Tenza D, Raposo G, Bonnerot C 2001 Secretory granules of mast cells accumulate mature and immature MHC class II molecules. J Cell Sci 114:323–334 Wang HW, Tedla N, Lloyd AR, Wakefield D, McNeil PH 1998 Mast cell activation and migration to lymph nodes during induction of an immune response in mice. J Clin Invest 102:1617–1626 Warbrick EV, Taylor AM, Botchkarev VA, Coleman JW 1995 Rat connective tissue-type mast cells express MHC class II: up-regulation by IFN-gamma. Cell Immunol 163:222–228 Wong GW, Foster PS, Yasuda S et al 2002 Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase gamma. TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an interleukin-13/interleukin-4 receptor alpha/signal transducer and activator of transcription (STAT) 6-dependent pathway. J Biol Chem 277:41906– 41915

Mastocytosis Dean D. Metcalfe Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Abstract. Systemic mast cell disorders in most instances appear to be clonal disorders of the mast cell and its progenitor. Symptoms result from a pathological release of mast cell mediators and a destructive mast cell infiltration. Cutaneous mastocytosis is most frequently seen in children and may regress. Systemic mastocytosis (SM) is a persistent disease. A somatic c-kit mutation at codon 816 is often detectable in haematopoietic cells. The clinical course of mastocytosis is variable, ranging from indolent to aggressive. Five categories of disease are recognized: Indolent SM, aggressive SM, SM with associated clonal haematological non-mast cell-lineage disease (AHNMD) and mast cell leukaemia (MCL). In SMAHNMD, additional genetic abnormalities have been reported. Patients with cutaneous or indolent systemic disease are treated symptomatically. Patients with aggressive disease are candidates for cytoreductive therapy. The use of ‘Kit-targeting’ tyrosine kinase inhibitors are best selected following a mutational analysis of c-kit. For instance, the D816V mutation appears to be associated with relative resistance against imatinib. However, imatinib has been used with success in patients with SM–hypereosinophilic syndrome (HES) and the FIPL1/PDGFRA fusion gene and in a patient with mastocytosis with a mutation outside of codon 816. The value of bone marrow transplantation remains under investigation. 2005 Mast cells and basophils: development, activation and roles in allergic/autoimmune disease. Wiley, Chichester (Novartis Foundation Symposium 271) p 232–249

The hallmark of mastocytosis is a pathological accumulation of mast cells at specific tissue locations such as in the skin, lymphoid tissues and bone marrow. As such, it is unique in representing heretofore the only primary disease identified that reflects abnormalities in the regulation of mast cell numbers. The study of this disease thus not only provides help to those with this disorder, but also provides insight into fundamentals in the understanding of mast cell biology and the role of mast cells in homeostasis and inflammatory disorders. Symptoms relating to mast cell degranulation may be observed in all categories of mastocytosis. These include episodic flushing with or without hypotension, dyspepsia, diarrhoea, abdominal pain and musculoskeletal pain. Patients who present with an associated non-mast cell clonal haematological disorder may also present with symptoms and findings that reflect the associated haematological disorder. 232

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Disease pathogenesis It is now known that human mast cells are derived from pluripotential CD34+ haematopoietic cells (Kirshenbaum et al 1991). Human mast cells may be cultured from CD34+ cells from blood or bone marrow when stem cell factor (SCF, also known as Kit ligand) is present in the culture media (Kirshenbaum et al 1992). No other growth factor or cytokine will substitute. In the human, SCF is encoded by a gene on chromosome 12 and is primarily produced by stromal cells (Anderson et al 1990). SCF may either be released as a soluble growth factor, or be expressed on the cell surface. Other cytokines in the presence of SCF modulate mast cell growth. For instance, interferon g inhibits mast cell outgrowth while interleukin (IL)6 facilitates mast cell proliferation. The receptor for SCF is Kit, a receptor tyrosine kinase. It is now accepted that activating mutations in c-kit are associated with an increase in mast cell numbers in most adult patients with mastocytosis, and in a subset of children with more severe disease. The first activating mutation identified in Kit in patients with clinical disease was Asp816 to Val (Nagata et al 1995). This mutation was first identified in peripheral blood mononuclear cells and subsequently identified in the skin and tissues of mastocytosis patients. Other activating mutations in c-kit at codon 816 have now also been identified including Asp 816Phe and Asp816Tyr (Longley et al 1999). The Asp816 Val mutation is detected in mast cells, T cells, B cells, and myelomonocytic cells in patients with mastocytosis; consistent with the conclusion that mastocytosis is a clonal haematopoietic stem cell disorder (Akin et al 2000a). Apart from D816V, other c-kit mutations and chromosomal defects have been reported to contribute to the development of mastocytosis in a few subjects (Table 1). Other defects, such as the FIPL1/PDGFRA fusion gene, have been associated with specific myeloid neoplasms and judged as reliable markers sufficient to define a certain subtype of patients with mastocytosis (Klion et al 2003). Thus, the presence of eosinophilia and FIPL1/PDGFRA in a patient with mastocytosis aids in the diagnosis of systemic mastocytosis with hypereosinophilic syndrome (SM-HES), a special variant of mastocytosis with an associated haematological disorder. Exactly how mutations in Kit facilitate the development of mastocytosis is not known. For instance, activating mutations in Kit do not reliably associate with increased cell proliferation in the presence or in the absence of SCF. What is known is that activating mutations in Kit promote the movement of mast cell progenitors towards SCF (Taylor et al 2001). This is one explanation for the collection of cells within mast cell lesions in bone marrow where the cells in such aggregates bear mutated Kit (Taylor et al 2004). The cells producing SCF which may attract other cells appear to be the mast cells themselves (Akin et al 2002). The B cells in such lesions are oligoclonal, arguing against such collections being clonal in nature

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TABLE 1 Gene defects, gene polymorphisms, and karyotype abnormalities associated with mastocytosis Finding

Disease association

% (estimated)

Gene defects c-kit D816V c-kit D816Y c-kit D816F c-kit D816H c-kit D820G c-kit V560G c-kit F522C c-kit E839K c-kit V530I c-kit K509I FIPL1/PDGFRA*

all variants of SM (and in some with CM) CM, SM, SM-AHNMD CM SM-AHNMD ASM SM SM CM SM-AML SM (familial type) SM-HES, SM with eosinophilia

>80%

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